Approach to infertility

APPROACH TO INFERTILITY
Presenter :
Dr. Varughese George
Moderator :
Dr. G. Mangala

Introduction
• Infertility - Failure of a couple to conceive/achieve a clinical
pregnancy after 12 months or more of regular unprotected
coitus.
•Subfertility - Any form of reduced fertility with prolonged
time of unwanted non-conception.
•Fecundability - Probability of achieving a pregnancy within one
menstrual cycle. (In a healthy young couple, it is 20 percent)
•Fecundity - Probability of achieving a livebirth within a single
cycle.

Introduction
• Types :
Primary infertility - those patients who have never
conceived.
Secondary infertility - indicates previous pregnancy
but failure to conceive subsequently.

Factors Essential for Conception
• Healthy spermatozoa should be deposited high in the
vagina at or near the cervix (male factor).
• Spermatozoa should undergo changes (capacitation,
acrosome reaction) and acquire motility (cervical factor).
• ™™Motile spermatozoa should ascend through the cervix
into the uterine cavity and the fallopian tubes.
• ™™There should be ovulation (ovarian factor).

Factors Essential for Conception
• ™™Fallopian tubes should be patent & oocyte should be
picked up by the fimbriated end of the tube(tubal factor).
• ™™Spermatozoa should fertilize oocyte at the ampulla of
the tube.
• Embryo should reach the uterine cavity after 3–4 days of
fertilization.
• ™™Endometrium should be receptive (by estrogen,
progesterone, IGF-l, cytokines, integrins) for
implantation, and the corpus luteum should function
adequately.

Epidemiology
• About 80% of couples achieve conception within their
first year of attempt.
• Another 10% will achieve within 2 years.
• Remaining 10% fail to conceive after 2 years.
• Many subfertile couples can achieve a pregnancy and
deliver a child.
• About 40% couples have primary infertility.
• 60% of couples have secondary infertility.

Causes of Infertility
• Male Infertility (30%)
• Female Infertility (30%)
• Both Male & Female Infertility(30%)
• Unexplained (10%)

Causes Of Male Infertility
Pre-testicular Testicular Post-testicular
Endocrine
• Gonadotropin deficiency
• Obesity
• Diabetes Mellitus
• Thyroid dysfunction
• Hyperprolactinemia
Psychosexual
–– Erectile dysfunction
–– Impotence
Drugs
–– Antihypertensives
–– Antipsychotics
Genetic
–– 47 XXY (Klinefelter’s
Syndrome)
–– Y chromosome deletions
–– Single gene mutation
• Immotile cilia (Kartagener)
syndrome
• Cryptorchidism
• Infection (mumps orchitis)
• Toxins: Drugs, smoking,
Radiation
• Varicocele
• Immunologic
• Sertoli-cell-only syndrome
• Primary testicular failure
• Oligoastheno-
teratozoospermia
Obstruction of efferent
duct
• Congenital
–– Absence of Vas
deferens
(Cystic fibrosis)
–– Young’s syndrome
• Acquired Infection
––Tuberculosis
––Gonorrhea
• Surgical
–– Herniorrhaphy
–– Bladder neck surgery
–– Vasectomy
• Others
–– Ejaculatory failure
––Retrograde ejaculation
–– Hypospadias

Approach to Male Infertility
History
 Type of lifestyle (heavy smoking, alcoholism)
 Sexual practices
 Erectile dysfunction and ejaculatory disorders
 Sexually transmitted diseases
 Surgery in genital area
 Drugs
 Any systemic illness.

Physical examination
 Testicular size
 Undescended testes
 Hypospadias
 Scrotal abnormalities (like varicocele)
 Body hair
 Facial hair

Semen Analysis
• Collected after about 3 days of sexual
abstinence.
• Obtained by masturbation.
• Collected in a clean, dry, sterile, and
leakproof wide-mouthed plastic container.
• Brought to laboratory within 1 hour of
collection.
• First portion  most concentrated &
contains the highest number of sperms.
• During transport to the laboratory, the
specimen should be kept as close to body
temperature as possible.

Semen Analysis
• During transport to laboratory, the specimen should
be kept as close to body temperature as possible.
• Specimen obtained near the testing site in an
adjoining room.
• Condom collection containing spermicidal agent is
not recommended.
• Collection of ejaculate post coitus interruptus is not
recommendated.
• Two semen specimens should be examined that are
collected 2-3 weeks apart

Semen Analysis
Test Result
Physical examination Time to liquefaction, viscosity, volume, pH,
color.
Microscopic examination Sperm count, vitality, motility, morphology,
and proportion of white cells
Immunologic analysis Antisperm antibodies
SpermMAR test
Immunobead test
Bacteriologic analysis Detection of infection
Sperm function tests Postcoital test, cervical mucus penetration
test, Hamster egg penetration assay,
hypoosmotic swelling of flagella, and
computer-assisted semen analysis

Semen Analysis
Physical Examination
Appearance :
Normal - Gray-white color, translucent
White turbidity - Presence of WBCs (infection)
Red color - RBCs (infection,malignancy)
Yellow color - Urine contamination, specimen collection
following prolonged abstinence and
medications.

Semen Analysis
Viscosity : Slightly viscous and easily drawn into a pipette.
Incompletely liquefied specimens will be clumped
and highly viscous.
Increased viscosity and incomplete liquefaction will
impede sperm motility.
Factors affecting semen viscosity
•Dehydration
•Infections of prostate/seminal vesicles
•Drugs

Semen Analysis
Volume: N= 1.5-5 ml
Decreased volume frequently associated with infertility
indicates improper functioning of one of the semen-
producing organs.
Low volume ejaculates are caused by 3 disorders :
• Ejaculatory disorders: Neurological diseases,Diabetes,Prior surgeries &
Medications; can cause retrograde ejaculation, aspermia or both.
• Genital tract obstructions : Prostate cysts,inflammatory disorders,Infections,
Scars from prior surgeries.
• Congenital anomalies : Seminal vesical aplasia(absence), congenital
absence of the vas deferens

Semen Analysis
pH : Normal pH is 7.2 to 8.0
Increased pH – infections within the reproductive tract.
decreased pH – increased prostatic fluid(acidic)
low semen volume.

Semen Analysis
Microscopic Examination
Sperm Motility
• 1st laboratory assessment of sperm function
• essential for penetration of cervical mucus, traveling
through the fallopian tube, and penetrating the
ovum.
• should be assessed as soon as possible after
liquefaction within 1 hour following ejaculation, to
limit the deleterious effects of dehydration, pH or
changes in temperature on motility

Semen Analysis
Sperm Motility
• A drop of semen is placed on a glass slide, covered
with a coverslip that is then ringed with petroleum
jelly to prevent dehydration, and examined under
40× objective.
• At least 200 spermatozoa are counted in several
different microscopic fields.

Semen Analysis
Sperm Motility
Result is expressed as a percentage of
• Progressive motility (PR): spermatozoa moving actively,
either linearly or in a large circle, regardless of speed.
• Non-progressive motility (NPR): all other patterns of
motility with an absence of progression
• swimming in small circles
• the flagellar force hardly displacing the head, or
• when only a flagellar beat can be observed.
• Immotility (IM): no movement
e.g.

Semen Analysis
Sperm Viability
• Decreased sperm viability may be suspected when a
specimen has a normal sperm concentration with
markedly decreased motility.
• Viable sperm cells will not take up the eosin Y stain,
whereas non-viable cells will be stained pink-red.

Semen Analysis
Microscopic Examination - Sperm Viability
Method
1. Mix one drop of semen with 1 drop of eosin-nigrosin
solution and incubate for 30 seconds.
2. A smear is made from a drop placed on a glass slide.
3. The smear is air-dried and examined under oil
immersion objective

Semen Analysis
Method
4. At least 200 spermatozoa are examined
White sperms are classified as live or viable Red sperms are classified as dead or non-viable

Semen Analysis
Method
5. The result is expressed as a proportion of viable sperms
against non-viable as an integer percentage.
58 % or more of sperms are normally live or viable.

Semen Analysis
Sperm Count
• Sperm count is done after liquefaction in a counting
chamber following dilution and the total number of
spermatozoa is reported in millions/ml (106 /ml)
• The amount of the dilution and the number of
squares counted varies among laboratories.

Semen Analysis
Microscopic Examination - Sperm Count
Method
1. Semen is diluted 1:20 with sodium bicaronate-
formalin diluting fluid
(1ml liquified semen in graduated tube and fill with
diluting fluid to 20ml mark & mix well)
2. Place a coverslip over Neubauer counting chamber
filled with well mixed diluted semen sample.
3. Spematozoa is counted in 4 large corner squares.

Semen Analysis
Method
4. Sperm count per ml is calculated as follows:

Semen Analysis
Method
5. Normal sperm count is ≥ 15 million/ml. Sperm
count < 15 million/ml may be associated with
infertility in males.

Semen Analysis
Microscopic Examination - Sperm Morphology

Semen Analysis
Sperm Morphology

Semen Analysis
Sperm Morphology
• Normally, > 4% of spermatozoa should show normal
morphology (WHO, 2010)
• Defects in morphology that are associated with infertility
in males include :
– defective mid-piece (causes reduced motility)
– incomplete or absent acrosome (causes inability to
penetrate the ovum)
– giant head (defective DNA condensation)

Microscopic Examination - Abnormal Sperm Morphology
Teratozoospermia, teratospermia or abnormal sperm
morphology is a semen alteration in which a large percentage of
sperm have an abnormal shape.
1. ABNORMAL SPERM HAVE LARGE OR DEFORMED HEADS

2. THE MIDPIECE MAY BE DEFECTIVE

• CROOKED OR DOUBLE TAILS

Test Result
Volume ≥ 1.5 ml
pH ≥ 7.2
Sperm concentration ≥15 million/ml
Total sperm count per ejaculate 39 million
Morphology ≥ 4.0% sperms with normal morphology
Vitality ≥ 58% live forms
White blood cells ≤ 1 million/ml
Motility within 1 hour of ejaculation
Total motility (PR + NPR)
Progressive motility (PR)
≥ 40% rapidly progressive
≥32% progressive
Mixed antiglobulin reaction (MAR) test <50% motile sperms with adherent particles
Immunobead test <50% motile sperms with adherent particles
Normal values of semen analysis (World Health Organization,2010)

Test Result
Total fructose (seminal vesicle marker) ≥13 μmol/ejaculate
Total zinc (Prostate marker) ≥2.4 μmol/ejaculate
Total acid phosphatase (Prostate marker) ≥200 U/ejaculate
Total citric acid (Prostate marker) ≥52 μmol/ejaculate
α-glucosidase (Epididymis marker) ≥20 mU/ejaculate
Carnitine (Epididymis marker) 0.8-2.9 μmol/ejaculate
Biochemical variables of semen analysis (World Health Organization,2010)

Biochemical analysis : Fructose test
1. Prepare reagent (50 mg resorcinol dissolved in 33 mL
concentrated HCl; diluted upto 100 mL with distilled water).
2. Mix 0.5 ml semen with 5 mL reagent.
3. Heat and bring it boil.
4. Observe for orange-red colored ppt within 30 seconds – Fr+
 Absence of fructose  obstructed or absent vas deferens.
 In azoospermia,
 if fructose is absent  obstruction of ejaculatory ducts or
absence of vas deferens
 if fructose is present  failure of testes to produce sperm.

Terminology Inference
Normozoospermia All semen parameters normal
Oligozoospermia Sperm concentration < 15 million/ml
mild to moderate: 5-15 million/ml
severe: < 5 million/ml)
Azoospermia Absence of motile/viable sperm in the
semen.
Aspermia Absence of ejaculate/semen
Asthenozoospermia Reduced sperm motility
<40% of sperms show total( progressive and
non-progressive) motility
or
<32% sperms showing progressive motility.
Teratozoospermia Spermatozoa with reduced proportion of
normal morphology (< 4% or increased
proportion of abnormal forms)
Leukocytospermia >1 million white blood cells/ml of semen
Oligoasthenoteratozoospermia All sperm variables are abnormal
Necrozoospermia All sperms are non-motile or non-viable

Semen Analysis - Evaluation of azoospermia.

Semen Analysis - Evaluation of low semen volume

Chromosomal analysis
Klinefelter’s syndrome (e.g. XXY karyotype)
 Deletion in Y chromosome
 Autosomal Robertsonian translocation
 Cystic fibrosis carrier state if bilateral congenital absence of
vas deferens is present.

• If semen analysis parameters are normal (according to
WHO criteria), further testing is not required.
• Further andrological tests are indicated only
– if at least two semen examinations (done 1 month apart) are
abnormal.
• In patients with abnormal semen analysis, the further
basic laboratory tests done are
• estimation of serum luteinizing hormone, follicle
• stimulating hormone and testosterone.

Hormonal studies
 Measurement of FSH, LH and testosterone to detect
hormonal abnormalities causing testicular failure.

Testicular biopsy
• Indicated when
differentiation between
obstructive and non-
obstructive azoospermia
is not evident (i.e. normal
FSH and normal testicular
volume).
• The biopsy material is to
be sent in
Zenker’s/Bouin’s solution
preferred over formalin
saline.
• Testicular tissues may be
cryopreserved for future
use in IVF/ICSI.

Testicular biopsy
• Adequate testicular biopsy for infertility should be about
the size of a grain of rice (3 mm) in any one dimension,
and will contain 3-5 lobules, or 9-20 seminiferous
tubules with intervening septa .
• One sample per testis is usually adequate, but it may be
necessary to obtain bilateral specimens as the nature
and/or stage of various lesions may differ from one testis
to the other.
• This sample can be obtained from the portion opposite
the rete testes via a 4-5 mm incision in the tunica
albuginea. This incision will cause a small herniation of
the parenchyma, allowing easy access to the testis.

Testicular biopsy
Biopsy specimens from infertile men with
azoospermia usually show one of the
following conditions:
• Germ cell aplasia (Sertoli cell-only
syndrome) (29%)
– tubules populated by only Sertoli cells,
measuring 100 –150 µm in diameter.
– tubular basement membrane
thickening.
– germ cells are completely absent.
– Leydig cells are usually normal but on
occasion are found to be reduced in
size and number.

Testicular biopsy
• Normal spermatogenesis (27%)
– usually associated with bilateral
obstruction as seen in Young’s Syndrome
or testicular blockage.

Testicular biopsy
• Spermatocytic arrest (26%)
– halt of the maturation sequence, usually
at the stage of the primary
spermatocyte.(presumably at the end of
the meiotic prophase at late pachytene)
– no spermatids or spermatozoa are
present despite the presence of
abundant cells in division.
– Leydig cells are normal.
• Generalized fibrosis (18%).

Testicular biopsy
Biopsy specimens from infertile men with oligozoospermia
usually show the following microscopic patterns:
• Incomplete spermatocytic arrest
– some tubules normal, some with arrest.
• Regional or incomplete fibrosis
• Spermatogenic hypoplasia
– tubules with reduced number of germ cells that are also
disordered.
• Tubular hyalinization
– includes Klinefelter syndrome(small diameter tubules
with thickened basement membrane and increased
Leydig cells).

Testicular biopsy
Biopsy specimens from infertile men with oligozoospermia
usually show the following microscopic patterns:
• Mixed atrophy
– synchronous occurrence of seminiferous tubules with
germ cells and tubules with Sertoli cells only.
• Normal spermatogenesis
– associated with duct obstruction.
• Sloughing and disorganization
– lumina contain desquamated immature cells, disordered
spermatogenesis (non-specific, can be seen in
hypoplasia, duct obstruction or mechanical damage/
artifact to specimen)

OBSTRUCTIVE AZOOSPERMIA HYPOSPERMATOGENESIS
SPERMATOGONIA ARREST
PRIMARY SPERMATOCYTE ARREST

SECONDARY SPERMATOCYTE ARREST SERTOLI CELL ONLY
SPERMATID ARREST TUBULAR FIBROSIS

Testicular biopsy
Histological grading of seminiferous tubules
(According to the modified Johnsen scoring)
Score Interpretation
1 No seminiferous epithelium
2 No germinal cells, Sertoli cells only
3 Spermatogonia only
4 No spermatozoa or spermatids, few spermatocytes
5 No spermatozoa or spermatids, many spermatocytes
6 No spermatozoa, no late spermatids, few early spermatids
7 No spermatozoa, no late spermatids, many early spermatids
8 Less than five spermatozoa per tubule, few late spermatids
9 Slightly impaired spermatogenesis, many late spermatids, disorganized
epithelium
10 Complete spermatogenesis with many spermatozoa present

Immunologic tests
• Antisperm antibodies (ASA) detected in semen, cervical
mucosa,or serum are considered a possible cause of
infertility.
• Two frequently used tests to detect the presence of
antibody- coated sperm are
1. Sperm Mixed Antiglobulin Reaction (MAR) tests:
– Detects both IgG and IgA antibodies against sperm
surface in semen sample.
– The tests requires fresh and unwashed semen.
2. Immunobead test :
– Detects the presence of IgG, IgM, and IgA antibodies
– Demonstrates autoantibodies are affecting specific
areas of the sperm (head, neck, or tail)

Immunologic tests
Sperm Mixed Antiglobulin Reaction (MAR) tests :
1. Direct Sperm MAR IgG test -
1 drop semen + IgG coated latex particles + anti-
human Ig on a glass slide
ASA +ve  IgG coated latex particles binds with IgG on
spermatozoa.

Immunologic tests
2. Direct Sperm MAR IgA test -
1 drop semen + IgA coated latex particles
ASA +ve  The latex particles will bind to
spermatozoa.

Immunologic tests
3. Indirect Sperm MAR tests –
• Serum/fluid without spermatozoa is tested for the
presence of ASA.
• Antibodies are bound to donor spermatozoa which are
then mixed with the fluid to be analyzed.
• ASA are detected as per direct sperm MAR tests.
• ASA +ve if >50% of spermatozoa show attached latex
particles.

Immunologic tests
Immunobead tests :
• Sperm are mixed with
polyacrylamide beads known to
be coated with anti-IgG/anti-
IgM/anti-IgA.
• Microscopic examination of the
sperm will show the beads
attached to sperm at particular
areas.
>50% spermatozoa with attached
beads – Abnormal finding

Immunologic tests
Sperm Agglutination Tests
1. Antisperm Antibodies Assay : Gelatin Agglutination Test (GAT) (KIBRICK)
1. Heat inactivated serum samples diluted 1:4 or serially with Baker's buffer.
2. Fresh semen adjusted with Baker's buffer to 40 x 106 cells/ml and warmed
to 37° C.
3. Mix warmed sperm suspension with an equal part of a 10% gelatin solution
in Baker's buffer at 37° C.
4. 0.3 ml of each serum sample is warmed to 37° C in a serologic tube and
mixed gently with 0.3 ml of sperm-gelatin mixture.
5. Each mixture is then transferred to a 3 x 30 mm tube.
6. Tubes are incubated at 37° C and results recorded at 1 h and again at 2 h.
7. Each run should include a known positive serum and a known negative serum ctrl.

Immunologic tests
Sperm Agglutination Tests
2. Microscopic Sperm Agglutination Test – Franklin & Dukes’ Test
• Fresh semen of at least 50% motility is diluted with Baker's solution to 50 million
spermatozoa/ml
• 0.05 ml of this dilute semen is mixed with 0.5 ml of serum that has been diluted in
Baker's solution 1: 4 and inactivated at 56°C for 30 min.
• This mixture is incubated at 37° C.
• After 0.5, 1, 2, and 3 h, one drop is examined under microscope and motile sperms
are counted in 12 fields.
• Notes should be taken of the following parameters: no: of free spermatozoa, no: of
clumped spermatozoa, no: of clumps and type of clumps - head to head,
tail to tail, etc.
• The total number of clumped spermatozoa is expressed in ratio to the total number
of all motile spermatozoa.
• If more than 20% of cells are clumped, the test is said to be positive.

Immunologic tests
Sperm Immobilization Tests
Isojima’s Sperm Immobilization T est
1. Fresh semen diluted to 60 x 105 cells/ml in saline.
2. Heat-inactivated serum samples are diluted serially with saline.
3. Mix 0.025 ml of semen with 0.25 ml of serum and 0.05 ml of pretitered active
complement (human, rabbit, or guinea pig serum).
4. In a duplicate tube, mix 0.025 ml of serum and 0.05 ml of heat-inactivated
complement.
This is to determine toxic effects by noncomplement dependent factors.
5. In each run. a saline, a known negative serum, and a known positive serum should
be included as controls.
6. Incubate at 37° C for 60 min.
7. Examine sample under a microscope and visually determine the percentage of motile
spermatozoa.
The test is considered positive when the loss of sperm motility is at least twice as great
in the patient's serum as in the control serum.

Transrectal ultrasound
(TRUS)
• Visualizes the seminal
vesicles, prostate and
ejaculatory ducts
obstruction.
• Indications :
– Azoospermia or severe
oligospermia with a normal
testicular volume.
– Abnormal digital rectal
examination.
– Ejaculatory duct abnormality
– Genital abnormality
(cysts, dilatation or calcification).
(hypospadias)

Approach to Female Infertility

• Indirect
– ™Menstrual history
– ™Evaluation of peripheral or
end-organ changes
• Basal Body Temperature
• Cervical mucus study
• Vaginal cytology
• Hormone estimation
– Serum progesterone
– Serum LH
– Urine LH
– Serum estradiol
– Endometrial biopsy
– ™Sonography (TVS)
• Direct
– Laparoscopy (Gold standard)
• Conclusive
– Pregnancy
Tests for Ovulation

Basal body temperature
• There is “biphasic pattern” of temperature variation in
ovulatory cycle.
• Secondary to rise in progesterone output following ovulation.
• Progesterone is thermogenic.
• Increase in the production and secretion of norepinephrine
which is also thermogenic.
• The body temperature maintained throughout the first
half of the cycle is raised to 0.5° - 1°F (0.2°– 0.5°C)
following ovulation.

Cervical mucus study:
Fern test
• seen in estrogenic phase.
• Fern formation is seen
when cervical mucus is
spread on a glass slide
• disappears after the 21st
day of the cycle -
presumptive evidence of
corpus luteum activity.
Spinnbarkeit test
• Elastic cervical mucus
withstands stretching
upto distance of 10 cm.
• cervical mucus viscosity
increases & breaks when
stretched during
secretory phase.
• change in cervical mucus
show e/o ovulation.

Vaginal cytology:
– Karyopyknotic index (KI) is high during estrogenic
phase.
– The peak KI usually corresponds with time of
ovulation and may reach upto 50-85.
– Maturation index shifts to the left from the midcycle
to the mid second half of cycle due to the effect of
progesterone.
– A single PAP smear on day 25 or 26 of the cycle
reveals features of progesterone effect.

Hormonal Estimation
• Serum progesterone estimation is
done on day 8 and day 21 of a 28 day
cycle.
• Daily estimation of serum LH at mid
cycle can detect the LH surge which
coincides about 10–12 hours after
the LH peak.
• Ovulation usually occurs within 14–
26 hours of detection of urine LH
surge and almost always within 48
hours.
•
Progesterone level > 10 nmol/L is
a reliable evidence of ovulation if
cycles are regular
Serum estradiol attains the peak rise 24 hours prior to LH surge
and about 24–36 hours prior to ovulation.

Endometrial biopsy:
• Biopsy is to be done on 21st –23rd day of the cycle.
• Evidences of secretory activity of the endometrial
glands in the second half of the cycle.
• Predicts the functional integrity of the corpus luteum.
• Subnuclear vacuolation is the earliest evidence
appearing 36–48 hours following ovulation.
• The secretory changes are due to the action of
progesterone on the estrogen primed endometrium.

Sonography:
• Serial sonography (TVS) during mid-cycle can precisely
measure the Graafian follicle of 18–20 mm just prior to
ovulation.
• Aids in confirmation of ovulation following ovulation
induction, artificial insemination, and in vitro fertilization.
• Features of recent ovulation are collapsed follicle and
fluid in the pouch of Douglas.

Laparoscopy:
• Laparoscopic visualization of recent corpus luteum or
detection of the ovum from the aspirated peritoneal
fluid from the pouch of Douglas.
• The only direct evidence of ovulation.

Luteinized unruptured follicle (LUF) syndrome :
• Refers to an infertile woman with regular menses and
presumptive evidences of ovulation without release of
the ovum from the follicle (trapped ovum).
• Features of ovulation—formation of corpus luteum and
its stigma are absent.
• Often associated with pelvic endometriosis.
• Sonography : Persistence of echo-free dominant follicle
beyond 36 hours after LH peak.
• Laparoscopy : Failure to observe a stigma of ovulation.
• Ovarian biopsy : Conclusive proof is determination of
ovum amidst the structure of corpus luteum.

Tests to Determine the Cause of Anovulation
• Measurement of LH, FSH, and estradiol during days 2-6.
– All values are low in hypogonadotropic hypogonadism (hypothalamic or
pituitary failure).
• Measurement of TSH, prolactin, and testosterone if cycles
are irregular or absent:
– Increased TSH: Hypothyroidism
– Increased prolactin: Pituitary adenoma
– Increased testosterone: Polycystic ovarian disease(PCOD), congenital
adrenal hyperplasia
To differentiate between both conditions, ultrasound and estimation of
DHEA are done.
• Transvaginal ultrasonography:
– done for detection of PCOD.

Sperm Function Tests
A. Post coital (Sims-Huhner test)
B. Cervical mucous penetration test
C. Hamster egg penetration assay
D. Hypoosmotic swelling of flagella
E. Computer assisted semen analysis

Postcoital test (PCT) (Sims-Huhner test)
• Examination of cervical mucous post coitus can give an
idea about the quality of cervical mucous and ability
of the sperms to penetrate it .
• This test is scheduled close to ovulation(12-13th day) when
mucus is abundant.
• Cervical mucous is aspirated with a syringe 2-12 hours
post coitus.
• Gross and microsocopic examination is carried out.

Interpretation of Postcoital test (PCT)

Cervical Mucus Penetration Test

Hamster egg penetration assay

Hypoosmotic swelling of flagella

Computer assisted semen analysis
•Automated system measures various characteristics of the spermatozoa
•The semen specimen is placed on the stage of the microscope.
• This microscope has a high resolution video camera attached.
• The video camera feeds data into the computer where it undergoes analysis by software.
•A computerized semen analysis will give many more parameters that are useful to the
pathologist.
•More importantly, the results are accurate and reproducible.

Computer assisted semen analysis - sperm motility
Squiggly green lines =>individual sperm paths
Red dots => sperm that are not moving

Computer assisted sperm morphology
“Strict Morphology”
A normal percentage under Kruger’s criteria is 14%
The software measures individual sperm cells
and shapes

Investigations to Assess Tubal and Uterine Status
Insufflation test (Rubin’s test)
• Entry of air/CO2 into the peritoneal cavity when pushed
transcervically under pressure, gives e/o tubal patency.
• Done in postmenstrual phase at least 2 days after stoppage of
menstrual bleeding.
• It should not be done in the presence of pelvic infection.
• The patency of the tube is confirmed by :
• fall in the pressure when raised beyond 120 mm Hg.
• hissing sound heard on auscultation on either iliac fossa.
• shoulder pain experienced by the patient (irritation of the diaphragm by the air).
• False-negative findings due to cornual spasm.
• Test also cannot identify the side and site of the block in the
tube.

• Infectious disease:
– Endometrial biopsy for tuberculosis
– Test for chlamydial IgG antibodies
• Hysterosalpingography (HSG)
– Assesses shape of the uterine cavity.
– Blockage of fallopian tubes

• Hysterosalpingo-contrast
sonography
– Catheter is introduced into
the cervical canal.
– Echocontrast fluid is
introduced into the uterine
cavity
– Shape of the uterine cavity,
filling of fallopian tubes and
spillage of contrast fluid are
noted.

• Laparoscopy and dye
hydrotubation test with
hysteroscopy
– A cannula is inserted into the
cervix.
– Methylene blue dye is
introduced into the uterine
cavity.
– If tubes are patent, spillage of
the dye is observed from the
ends of both tubes.

To summarize
• Infertility is defined as a failure to conceive within 1 year
of regular unprotected coitus.
• Male factor is responsible in 30–40 percent, the female in
40–50 percent, both in 10 percent and unexplained in 10
percent.
• Common causes of male infertility include defective
spermatogenesis, obstruction of efferent duct, failure to
deposit sperm high in vagina and error in seminal fluid.
• Important causes of female infertility are tubal factor
(25–35%), ovulatory factor (20–25%) and endometriosis
(0–10%).

To summarize
• Investigation of the infertile couple should be started
after 1 year.
• The initial investigations of an infertile couple include:
semen analysis (Male factor), hysterosalpingography
(tubal factor), Basal body temperature and/or serum
progesterone (for evidence of ovulation).
• A thorough investigation following an abnormal
semen analysis, is needed to find out the specific
cause of male infertility.
• Tubal factor is assessed by Hysterosalpingogram and
laparoscopy

References
• Kawthalkar SM. Essentials of clinical Pathology. Jaypee Brothers, Medical
Publishers Pvt. Limited; 2018 Jul 31.
• World Health Organization. WHO laboratory manual for the examination
and processing of human semen.
• Lindsay TJ, Vitrikas KR. Evaluation and treatment of infertility. Women.
2015 Mar 1;100(8):26-7.
• Abdullah L, Bondagji N. Histopathological patterns of testicular biopsy in
male infertility: A retrospective study from a tertiary care center in the
western part of Saudi Arabia. Urology annals. 2011 Jan;3(1):19.
• Sikka SC, Hellstrom WJ. Current updates on laboratory techniques for the
diagnosis of male reproductive failure. Asian journal of andrology. 2016
May;18(3):392.
• Brugo-Olmedo S, Chillik C, Kopelman S. Definition and causes of infertility.
Reproductive biomedicine online. 2001 Jan 1;2(1):173-85.
• Anawalt BD. Approach to male infertility and induction of
spermatogenesis. The Journal of Clinical Endocrinology & Metabolism.
2013 Sep 1;98(9):3532-42.

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