INFERTILITY MANAGEMENT

AND THE ROLE OF SEMEN

ANALYSIS AND SPERM
FUNCTION TESTS
Dr Suryakant Hayatnagarkar
Cryo Cell India Pvt. Ltd
New Delhi
Cryo Cell

Cryo Cell

COMPOSITION OF SEMEN
Spermatozoa
Seminal Plasma containing secretions from
Testicular
Epididymal
Seminal Vesicular
Prostatic
Bulbourethral Glands

Cryo Cell

SAMPLE COLLECTION
Clear Instructions to the Patient
Patient should be reassured and counseled
about sample collection.
Sample collection should be in special private
room near Laboratory in most comfortable
atmosphere.
Collection container should be nontoxic
disposable plastic wide mouth container

Cryo Cell

SAMPLE COLLECTION

In case of inablity to masturbate sample

collection can be done in special condoms
without spermicidal jelly.
Sample collected at home should be brought in
warm conditions.
Coitus interreptus is to be avoided.
Abstinence Period between 2 to 5 days
Ensure Completeness of sample

Cryo Cell

INITIAL EVALUATION

Evaluate Immediately
Store in Incubator at

37oC
Detailed examination at
the end of one hour
Cryo Cell

INITIAL EVALUATION

Coagulation, Liquefaction and Viscosity

Semen Liquefies within 20-30 minutes.
Viscosity tested by transferring by pipette to
tube drop by drop.
Abnormal coagulation reflects
prostatic
dysfunction but is not a cause of infertility if
other parameters are normal

Cryo Cell

INITIAL EVALUATION
Appearance and Color
Normal color Grayish White
Opalescent
Abnormal colors yellow, red, pink
brownish, clear transparent
Cryo Cell

INITIAL EVALUATION
VOLUME

Cryo Cell

INITIAL EVALUATION
Volume
Normal Volume 2-6 ml
pH
Normal pH 7.2-8.0
Cryo Cell

INITIAL MICROSCOPIC
EVALUATION
10 micrtolitre well mixed sample is taken on

clean warm slide and covered with 22x22 mm

cover slip.

Examined after 5 minutes stabilization at 37 oC

Evaluation is made for initial count and

percentage of motility under phase contrast

microscope.

Cryo Cell

MOTILITY CLASSIFICATION SCHEME

WHO 1999

Class a

Rapid Progressive motility

Class b

Slow/sluggish progressive

Class c

Non-progressive motility

Class d

Non-motile

Cryo Cell

SPERM AGGLUTINATION

Cryo Cell

OTHER CELLULAR
COMPONENTS AND DEBRIS
Round Cells
Leukocytes, Immature germ cells
Epithelial Cells
Erythrocytes
Particulate Debris
Bacteria and Protozoa
Cryo Cell

OTHER CELLULAR
COMPONENTS AND DEBRIS

Cryo Cell

OTHER CELLULAR
COMPONENTS AND DEBRIS

Cryo Cell

Cryo Cell

SCHEME FOR RANKING

PARTICULATE DEBRIS
0
1
2
3
4
Cryo Cell

No Debris at all
Slight debris
Moderate
Heavy
Very heavy

Rare situation
Typical
Normal (?)
Abnormal
Abnormal

SPERM COUNTING

Improved Neubauer Chamber

Makler Chamber

CryoCell sperm Counting Chamber

Cryo Cell

NEUBAUER CHAMBER

Cryo Cell

MAKLER CHAMBER

Cryo Cell

Cryo Cell Sperm Counting

Chamber

Simple and Inexpensive Cryocell is easy to use and

gives rapid and accurate counts from undiluted specimen
Cryo Cell

CRYO CELL CHAMBER

Cryo Cell

CRYO CELL CHAMBER

Cryo Cell

CRYO CELL CHAMBER

Cryo Cell

ASSESSMENT OF
MORPHOLOGY

Wet Mount under Phase Contrast Microscope

Preparation of Smears
Staining
Hematoxylin-eosin
Papanicolaou
Leishman-Giemsa
Shorr

Cryo Cell

CRYO-MORPHO KIT
Material included with the Kit
Reagent A: red stain - 50ml in coplin jar
Reagent B: Blue stain - 50ml in coplin jar
Fixative 10 ml
(Replace the cap and inner plug with

dropper cap before first use.)

Cryo Cell

ASSESSMENT OF
MORPHOLOGY
Preparation
Ensure the fluid level is high enough to cover the area that

is to be stained. Fill two Coplin jar, or any other recipient

that can hold the slides, with tap water (for washing the
slides between the different dyes). If the tap water is
alkaline (pH > 7), then use distilled water for washing.
Use washed clean slides with frosted end for making
smears.
Cryo Cell

ASSESSMENT OF
MORPHOLOGY
Method
1. Allow a thin feathered-edge smear of fresh,

undiluted semen to air dry for about 5 minutes on a

warm plate at 37C.
2. Fix the smear by putting few drops of fixative on
horizontal slides so that the fixative covers all the
areas of smear. Allow the fixative to evaporate and let
the slides dry completely .
Cryo Cell

ASSESSMENT OF
MORPHOLOGY
Stain in stain A

using 3-5 dips of 1 second each.

Wash by dipping 7-10 times in fresh tap water.
Briefly drain excess water onto absorbent paper.
4. Stain in stain B using 7-8 dips of 1 second each.
Wash by dipping 7-10 times in fresh tap water.
Briefly drain excess water onto absorbent paper.
5. Allow smear to air dry.
Cryo Cell

ASSESSMENT OF
MORPHOLOGY
Observe staining under a light microscope

(100x) using oil immersion:

- acrosome = bluish pink
- nucleus = stained dark blue
- equatorial region = pale pink
- midpiece and tail = pink
Cryo Cell

ASSESSMENT OF
MORPHOLOGY
Interpretation of the results
Count at least 100 and preferably 200 spermatozoa and
classify them as either normal or abnormal, specifying
which defects are most common.
- Only include identifiable sperm cells in the count.
- The criteria for classifying sperm cells as either normal
or abnormal depends on the classification method used in
the lab.
Cryo Cell

ASSESSMENT OF
MORPHOLOGY
There is large variation in morphology of sperms
in normal individuals.
What to consider as typical normal morphology?
Studies on selection of spermatozoa in vivo in
female genital tract and in vitro studies have
shown uniform selection of spermatozoa and
helped in determining strict normal parameters
of morphology.
Cryo Cell

SPERM DIMENTIONS

Sperm Head
3-5 micron length, 2-3 micron width with
perfect oval shape
Mid-piece
7-8 micron length, 1 micron diameter,
straight, regular outline and aligned in
longitudinal axis of head
Tail
slender, uncoiled 45 microns in length

Cryo Cell

NORMAL SPERM
MORPHOLOGY

Cryo Cell

CLASSIFICATION OF SPERM
MORPHOLOGY
HEAD DEFECTS

Cryo Cell

CLASSIFICATION OF SPERM
MORPHOLOGY
HEAD DEFECTS

Cryo Cell

CLASSIFICATION OF SPERM
MORPHOLOGY
NECK AND MIDPIECE DEFECTS

Cryo Cell

CLASSIFICATION OF SPERM
MORPHOLOGY
TAIL DEFECTS

Cryo Cell

TERATOZOOSPERMIC INDEX

Teratozoospermic Index =
Total number of Defects / number of
Spermatozoa with defects

Sperm Deformity Index =

Total Number of Defects / Number of
Spermatozoa

TZI of >1.6 is associated with low pregnancy rates

Cryo Cell

INTERPRETATION OF SEMEN
ANALYSIS RESULTS

Approaches for finding out minimum

standards for fertile semen.

Compare results of semen evaluations in groups of

fertile and infertile males.
Compare results in infertile couples in those
conceiving and those not conceiving during
treatment.
It is difficult to depend on single evaluation or
single
Cryo
Cell group of tests for predicting fertility

VARIABILITY IN SEMEN
PARAMETERS
Day to Day variation
More than 50 % variation in two analyses
Period of Abstinence
Counts increase with days of Abstinence
Quality declines with more than 7 days of
Abstinence

Cryo Cell

VARIABILITY IN SEMEN
PARAMETERS
Occupational Influences
Exposed to high temperatures
Exposed to hazardous chemicals and

radiation
Truck and lorry drivers
Jobs with very high stress levels
Cryo Cell

VARIABILITY IN SEMEN
PARAMETERS
Influence of Illness
Febrile Illness
Common Cold
Viral Fevers
Cryo Cell

REFERENCE VALUES SEMEN

PAREMETERS

VOLUME
pH
Sperm concentration
Motility
Vitality
Round cells
Immunobead test

Cryo Cell

1.5 ml or more
7.2 or more
15 million/ml
50 % motile
50 % or more
less than 1 million/ml
< 50 % with bead bound

NOMENCLATURE
Oligospermia
Azoospermia
Teratozoospermia
Asthenospermia
Oligo-terato-asthenospermia
Aspermia
Cryo Cell

INTERPRETATION OF SEMEN
ANALYSIS RESULTS
HOW TO INTERPRET PREPARED
SEMEN ANALYSIS REPORT?
WHAT IS NORMAL?
IS ABNORMAL SUBFERTILE OR
INFERTILE?
HOW SOME SUBFERTILE ALSO
CONCEIVE?
Cryo Cell

INTERPRETATION OF SEMEN
ANALYSIS RESULTS
It is very easy to determine what is
normal by testing large number of
samples of proven fertility but what
minimum parameters are essential for
fertility remains unanswered.
Cryo Cell

SPERM CONCENTRATION
Receives undue attention of clinicians as well as patients as a
sole criterion of potential fertility .
Low sperm count is very easy way out to explain failure of
conception.
Remains unambiguous due to quantitative measurement but
very difficult to say what minimum number required for
fertilization.
Studies indicate even persons with 5 million sperms have
fathered when other parameters are normal.
Cryo Cell

SPERM MOTILITY
Most problematic feature to interpret.

No of motile sperms v/s average speed?

Motility is important for transport from
vagina to uterus and then in the fallopian
tubes
Marginal role in reaching ovum in the
fallopian tubes.
Cryo Cell

SPERM MORPHOLOGY
PRIMARY ABNORMALITIES

Introduced at the time of production

Macrozoospermia, macrozoospermia
Tail defects

SECONDARY ABNORMALITIES

Introduced during transport from testes to emission

Neck abnormalities and tail abnormalities
Cryo Cell

NUCLEATED CELLS
WHITE BLOOD CELLS

Produce toxins
Phagocytic action
GERMINAL CELLS

Precursor cells released during production

due to sloughing of cells from germinal

epithelium
Cryo Cell

SPERM FUNCTION
TESTS

Cryo Cell

SPERM MEMBRANE FUNCTION

Sperm Vitality Test

Eosin stain does not penetrate the intact cells
Penetrates cells with broken and damaged

membranes.
Nigrosine gives dark background so that
evaluation becomes easy.
Dead or damaged cells are stained various shades
of pink and normal vital cells are white.
Cryo Cell

Cryo-Vitality EN
MATERIAL INCLUDED IN THE KIT
Cryo-Vitality EN

1 ml
The stain is sufficient for doing at least 10-20 tests
Before first use replace the cap and inner plug with
the dropper provided in the pack so that dropper
can be used to remove reagent for tests.
Cryo Cell

Cryo-Vitality EN
PROCEDURE
1. Mix equal volumes of well-liquefied semen and
Cryo-Vitality EN (small drops) on a pre-cleaned
microscopic slide. Allow about 30 seconds for staining.
2 Make smear on the slide using other slide as spreader.
Smear should be thin enough for allowing proper
visualization. Thick smears are useless for evaluation.

Cryo Cell

Cryo-Vitality EN
3

Allow air-drying and preferably mounting with

DPX or equivalent mountant as soon as possible.
These slides can be stored at ambient temperature
indefinitely.
4 At least 100 or preferably 200 spermatozoa are
examined at a magnification of 1000x or 1250x under
oil immersion using high quality non-phase-contrast
objective and correctly adjusted bright-field optics.
Cryo Cell

SPERM MEMBRANE FUNCTION

Hypo-osmotic Swelling Test Principle

The HOS test is based on the ability of live spermatozoa to

withstand moderate hypo-osmotic stress. Dead spermatozoa whose
plasma membranes are no longer intact do not show swelling. Also
the dead spermatozoa with intact plasma membranes have no osmoregulatory mechanism active and show uncontrolled swelling
resulting in the rupture of their over distended plasma membranes.
The spermatozoa that show controlled swelling under test conditions
is considered the good fraction with low osmotic fragility.

Cryo Cell

SPERM MEMBRANE FUNCTION

Reagents

Cryo-HOS reagent

1.0 ml in ampoule
150 mOsm solution of Sodium Citrate and
Fructose
Parafilm sheets
Cryo Cell

SPERM MEMBRANE FUNCTION

Method:
Transfer the number of ampoules required to run the batch

of tests to incubator at 37oC. Allow to stabilize temperature

for 10-15 minutes
Mix 0.1 ml of liquefied semen with 1.0 ml reagent in
ampoule and cover with parafilm
Incubate for 30 minutes at 37oC. Remix the solution and
transfer one drop of sperm suspension to clean microscope
slide and mount using 22x22 mm cover-slip.
Cryo Cell

SPERM MEMBRANE FUNCTION

Method:
Transfer the number of ampoules required to run the batch

of tests to incubator at 37oC. Allow to stabilize temperature

for 10-15 minutes
Mix 0.1 ml of liquefied semen with 1.0 ml reagent in
ampoule and cover with parafilm
Incubate for 30 minutes at 37oC. Remix the solution and
transfer one drop of sperm suspension to clean microscope
slide and mount using 22x22 mm cover-slip.
Cryo Cell

SPERM MEMBRANE FUNCTION

Method:
Transfer the number of ampoules required to run the batch

of tests to incubator at 37oC. Allow to stabilize temperature

for 10-15 minutes
Mix 0.1 ml of liquefied semen with 1.0 ml reagent in
ampoule and cover with parafilm
Incubate for 30 minutes at 37oC. Remix the solution and
transfer one drop of sperm suspension to clean microscope
slide and mount using 22x22 mm cover-slip.
Cryo Cell

SPERM MEMBRANE FUNCTION

Method:

Examine using preferably phase contrast microscope at

magnification of 400x and count at least 100 (preferably
200) spermatozoa. (If phase contrast microscope is not
available normal binocular microscope with reduced lighting
can be used equally) Each spermatozoon is scored as either
normal or showing swelling of the tail region.
Cryo Cell

SPERM MEMBRANE FUNCTION

Results

The result of the test is expressed as the percent swollen

cells, equivalent to the proportion of osmotically competent

spermatozoa. Osmotically incompetent and dead
spermatozoa burst open and show straight tails.

Cryo Cell

HYPO-OSMOTIC SWELLING
TEST

Cryo Cell

HYPO-OSMOTIC SWELLING
TEST

Cryo Cell

TESTS OF SPERM MEMBRANE

BINDING, CAPACITATION AND
PENETRATION

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Hyper-activated Motility

Hemi-Zona Assay

Zona free hamster oocyte test

ACROSOME DETECTION AND

REACTION TESTS

Triple Staining

Fluorescent dyes binding

Various Lectins, Chlortetracycline,

Antisera etc.

Cryo Cell

Acrosin Measurement

Gelatin Film Lysis test

ACROSOME INTACTNESS TEST

Material included with the Kit

Incubation reagent 0.5 ml x 10

ampoules
Pre-coated Gelatin Film Slides 10
no.
Cryo Cell

ACROSOME INTACTNESS TEST

Method
Open the diluents ampoule.
Add 100 l of well mixed semen sample.

Shake a little and incubate the mixture for 5

minutes at room temperature.
Apply one small drop of mixture to the precoated gelatin film slide and spread smoothly
and evenly.
Cryo Cell

ACROSOME INTACTNESS TEST

Allow the fluid to dry partially and

then transfer to a moistened petri dish

(not touching the moistened tissue) and
incubate at 500 C for 30 minutes.
Examine halos around the sperm head
under microscope using high
magnification ( x40).
Cryo Cell

ACROSOME INTACTNESS TEST

Cryo Cell

ACROSOME INTACTNESS TEST

Interpretation:

Under microscope halos will be

seen around the sperm heads. The

sperm heads showing no halos are
considered negative for acrosomal
status and function.
Cryo Cell

ACROSOME DETECTION AND

REACTION TESTS

Cryo Cell

BIOCHEMICAL ASSAYS OF
SEMINAL PLASMA

PROSTATE
Zinc, Citric Acid, Acid Phosphatase

SEMINAL VESICLES
Fructose

EPIDIDYMIS
L-Carnitine, Neutral Glucosidase

Cryo Cell

SEMEN FRUCTOSE TEST

Simple biochemical test

Denotes intact seminal vesicle function

When present the levels directly

proportional to testosterone levels in
blood

Cryo Cell

SEMEN FRUCTOSE TEST

CRYO-FRUCTO~QUAL

Reagents :
Cryo-Fructo-Qual reagent
20 ml
Fructose Standard 5.0 mg/ml
1 ml

Cryo Cell

SEMEN FRUCTOSE TEST

Method :

1.

Take 2 ml Cryo-Fructo-Qual reagent 2 test

tubes. Label one as test(T) and other and
standard(S).
2 Add 0.2 ml standard in S tube and 0.2 ml
semen sample in T tube. Mix well.
3 .
Cryo Cell

SEMEN FRUCTOSE TEST

Keep both the tubes in boiling water

bath which is already boiling for one

minute. Cool by keeping in cold water.
4 Examine the color of both the tubes.
Standard will show pink-red color

Cryo Cell

SEMEN FRUCTOSE TEST

Keep both the tubes in boiling water

bath which is already boiling for one

minute. Cool by keeping in cold water.
4 Examine the color of both the tubes.
Standard will show pink-red color

Cryo Cell

SEMEN FRUCTOSE TEST

Interpetation:
Samples showsing similar or more color

than the Standard are interpreted as

positive for fructose. Colorless or very
faint are interpreted as negative for
fructose.
Cryo Cell

SEMEN FRUCTOSE
QUANTITATION TEST

Cryo Cell

SEMEN FRUCTOSE TEST

CRYO-FRUCTO~QUANT Reagents :
Precipitating Reagent, 10 % TCA for

precipitation of semen proteins. 0.9 ml x10

microcaps.
Color Reagent, Indole-3-acetic acid reagent.
0.5 ml
Calibrator, Fructose Standard solution 500
mg/dl.
Cryo Cell

SEMEN FRUCTOSE TEST

CRYO-FRUCTO~QUANT Method :
Mark one micrcap for calibrator and one for each

sample.
Pipette 0.1 ml of calibrator and 0.1 ml well mixed
semen sample in each labeled microcap. Mix well
and allow 10 minutes for precipitation.
Centrifuge all sample microcaps at 1500 RPM for 5
minutes to settle the precipitated proteins and obtain
clear supernatant.
Cryo Cell

SEMEN FRUCTOSE TEST

Label another set of empty microcaps and transfer

20 micro-liters of diluted calibrator and sample

supernatant to appropriately labeled microcaps.
Add 100 micro-liters color reagent in all the tubes.
Add 1 ml concentrated Hydrochloric acid to each
tube and mix well.
Incubate at 370C for 75 minutes. Keep all the
microcaps closed.
Cryo Cell

SEMEN FRUCTOSE TEST

Read absorbance at 520 nanometer or

equivalent filter on colorimeter.

Calculate using following formula
Concentration of test = mg/dl

Cryo Cell

SEMEN FRUCTOSE TEST

Assay levels and interpretation:
Concentration of fructose in semen ranges

from 63 to 500 mg/dl (3.5 to 28 mmol/l). It

must be remembered that as sample ages;
the fructose level will fall due to utilization
of fructose by spermatozoa. The more sperm
that is present in the ejaculate, the greater
the fall in fructose concentration.
Cryo Cell

CRYO-NCD KIT
Material included with the Kit

NCD Incubation reagent 0.5 ml x 10 ampoules

NCD Dry reagent disks

10 nos
Fixative and stain reagent 1 ml
Parafilm sheets
Cryo Cell

CRYO-NCD KIT
Method:
Open one ampoule of NCD Incubation reagent.

Add one disk of NCD Dry reagent to the pipette

reagent, cover with piece of parafilm and shake
to let the disk elute.
Incubate the vial at 50oC for 5-10 minutes to
achieve equilibration at this temperature.
Cryo Cell

CRYO-NCD KIT
Add 100 microlitre of well mixed semen sample

to the incubation mixture and incubate further

for 5 minutes exactly.
At the end of incubation time add one drop of
fixative and stain reagent to the incubation
mixture.

Cryo Cell

CRYO-NCD KIT
Mix well and take a small drop on slide and

place a 22x22 mm cover-slip.

Examine under low power and then high power
of microscope. The cells showing swelling of
nucleus with uniform chromatin are considered
positive and the cells showing intact nucleus are
considered negative NCD.
Cryo Cell

CRYO-NCD KIT
Results

The result of the test is expressed as the percent

swollen cells or NCD reacted cells to total sperms.

More than 70 % sperms in normal samples show
NCD positivity. Less than 70 % NCD positivity is
associated with reduced fertility of the sperm.
Cryo Cell

CRYO-NCD KIT

Cryo Cell

ANTI-SPERM ANTIBODY TEST

ELISA TESTS

IMMUNOBEAD TEST
IgG and IgA types direct and indirect tests

MIXED ANTIGLOBULIN REACTION

TEST

Cryo Cell

CERVICAL MUCUS TEST

Cryo Cell

SPERM CERVICAL MUCUS

INTERACTION

Cryo Cell

COPMUTER ASSISTED
SEMEN ANALYSIS

Cryo Cell

THANK YOU
Cryo Cell