Interrutional Fertiity &

Research Cantre

SEMEN ANALYSIS

DR.RENUKADEVI
SENIOR EMBRYOLOGIST
ARC International Fertility & Research
 Centre

International Fertility &Introduction

Research Centre

A semen analysis measures the amount of semen a m

produces and determines the number and quality of sperm
the semen sample.

A semen analysis is usually on e of the first test done to

determine whether a man has problem fathering a c
(infertility)

A problem with the semen or sperm affects more than one-t

of the couples who are unable to have children (infertile)

Semen Analysis
 SA are mainly carried out to determine whether:-
A man has a reproductive problem that is causing infertility.
A vasectomy or vasectomy reversal has been successful
SA is the singular test forfertility in male that can provide
information on
·sperm production.
。patency of the male ducts.
the function of the accessory glands.
。and ejaculative function.
Since semen samples may vary from day to day, 2 or 3 samples may be
evaluated within a 3-6 month period for accurate testing.
A semen analysis to test the effectiveness of a vasectomy is usually
done 6 weeks after the vasectomy
 Semen collection
Masturbation, directing the semen into a clean sample cup. Do not use
a lubricant which can kill sperms

Coitus interruptus - withdrawing the penis from the partner just before
ejaculating follow by ejaculating into a clean sample cup.

Coitus - by using a condom. A special (silicon) condom that does not

contain any substance that kills sperm (spermicide).After ejaculation,
carefully remove the condom from the penis.Tie a knot in the open
end of the condom and place it in a container that can be sealed in
case the condom leaks or breaks.Ordinary condoms should not be
used since they usually contain spermicides

Assisted ejaculation-clectro-ejaculation used in paralegics

 SEMEN ANALYSIS
Once after collecting the semen sample from the
patient,eceiving time is noted down.

The sample has to be liquefied for 20-30mins at 37c by placing

the sample in heating block. (Avoid crystallization by leaving
the sample stationary for a too long time).

After 20-30mins of liquefaction, mix the sample homogencously by

using transfer pipette.

Sample volume, viscosity and pH should be noted.

 Semen Collection
Good and reliable SA results starts from semen collection, preferably by
masturbation
Abstinence days 2-5
Pass urine
Wash hands with soap,dry
Collect the entire sample into the
wide mouth sterile
container,70% of sperms is in the
first part of the ejaculate
Keep the sample at body
temperature, no sunlight
Deliver the sample within one hour
of ejaculation

Parameters (WHO criteria 2010)

Parameter Lower Reference Limit

Semen volume (ml) 1.5

Sperm concentration (10%/ml) 15

Total sperm number (106/ejaculate) 39

Progressive motility (PR,%) 32

Total motility (PR +NP,%) 40

Vitality (live sperms, %) 58

Sperm morphology (NF,%) 4

pH*

Leucocyte*(106/ml) <1

MAR/Immunobead test*(%) <50

Parameters agreed on consensus

 Terminologies of Semen Analysis
Oligospermia-sperm concentration <15 million/ml
Asthenozoospermia-<40% grade (PR+NP) or< 3:2 PR%

Teratozoospermia -<4% spermatozoa

OAT=Oligo-astheno-teratozoospermia

Azoospermia-no spermatozoa in semen

Polyzoospermia-++high sperm concentration,>200

Hypospermia-semen volumc<1.5ml
Hyperspermia-semen volume>4.5ml

Aspermia-no semen volume

Pyospermia-leukocytes present in semen,>IM/ml

Hematospermia-red blood cell present in semen

Necrozoospermia-"dead" sperm
 Terminologies of Semen Analysis
Oligospermia-sperm concentration <15 million/ml
Asthenozoospermia-<40% grade (PR+NP) or< 3:2 PR%

Teratozoospermia -<4% spermatozoa

OAT=Oligo-astheno-teratozoospermia

Azoospermia-no spermatozoa in semen

Polyzoospermia-++high sperm concentration,>200

Hypospermia-semen volumc<1.5ml
Hyperspermia-semen volume>4.5ml

Aspermia-no semen volume

Pyospermia-leukocytes present in semen,>IM/ml

Hematospermia-red blood cell present in semen

Necrozoospermia-"dead" sperm

Macroscopic Examination
 WHO criteria 2010 Description

Appearance Normal: Whitish to grey opalescent

Yellow (urine,jaundice); Pink/Reddish/Brown (RBCs)

Liquefaction Normal: 15-30 minutes after collection

Lumpy >60 min -sperms may be trapped in unliquefied

jelly; maybe sign of prostatic infection, lack of prostatic
protease

Viscosity Normal Smooth and watery

Abnormal-, thick with long threads (21G needle). High

viscosity impede sperm movements

MIX SEMEN THOROUGHLY BEFORE ANALYSIS -USE A

Macroscopic Examination
Volume Normal: 1.5 ml per ejaculation

Low volume (<1ml) reflect a problem with the seminal

vesicles and prostate-a block, retrograde ejaculation,

infection or lack of androgen.

Low semen volume cannot neutralize vaginal acidity
High semen volume dilute sperms/active infection

pH Normal: =/>7.2 (alkaline)

Acidic pH (<7.0) in a low volume & densitysample

indicates-congenital bilateral absence of vas deferens (in
which seminal vesicles are also poorly developed)and
ejaculatory duct obstruction. pH increases with time as
natural buffering capacity of semen decreases -therefore
high ph is not clinically useful
Macroscopic examination

Lipufaction:

·Liquefaction is the breakdown of the gel portion of the seminal plasma-the enzymes for this
are in the prostatic fluid
·Semen is normally ejaculated as a coagulum and liquefied within 20 minutes after
ejaculation(37℃).

·Non liquefied semen is a fairly rare occurrence, may indicate prostatic dysfunction,and should be
noted.
·Aspirate the specimen into plastic pasture pipette and observe it
If homogencous and quite watery" Liquefaction is complete
If heterogeneous mixture Liquefactionis not complete

Note: Normal liquefied semen samples may contain jelly-like granules (gelatinous bodies) which
do not liquefy.
If after 2 hours the specimen has not liquified proteolytic enzymes such as alpha-chymotrypsin
may be added to allow the rest of the analysis to be perfonned.
Macroscopic examination

Vianity:
Measured by drawing and releasing semen from a pipet and notingviscosity using a
subjective scale.
Normal Sample Leaves the Pipette In small discrete drops.
(+) The drop will form a thread about 2 cm long.
(++) The drop will form a thread more than 2 cm long
(+++) No drops formed,shows a continuous filament.
(++++) Not aspirated into the plastic pasture pipette.

If viscosity is high repeat gently passage of specimen into pipette several times.
If viscosity is very high add equal volume of sperm media following by repeating pipetting.

Semen Analysis
SA are mainly carried out to determine whether:-
 A man has a reproductive problem that is causing infertility.
A vasectomy or vasectomy reversal has been successful
SA is the singular test for fertility in male that can provide information
on
sperm production.
°patency of the male ducts.
·the function of the accessory glands.
。and ejaculative function.
Since semen samples may vary from day today, 2 or 3 samples may be
evaluated within a 3-6 month period for accurate testing.
A semen analysis to test the effectiveness of a vasectomy is usually
done 6 weeks after the vasectomy
Macroscopic examination

Color
Normal Grey-opalescent
Low sperm Count Less Opaque
RBCs present Red-brown or pinkish
Drugs or vitamins Yellow

-The presence of gross macroscopic particles or debris should be noted.pHt

-Secretions from the prostate and seminal vesicles contribute to seminal pH.
·Measured using litmus paper.

- The pH of the semen is normally (7.2-7.8)

-Abnormal pH may be indicative of secondary sex gland

dysfunction).

Microscopic Examination
SPERM COUNT
A microscope objective of 20x should be used for counting.

Place a drop of the sample into Makler chamber and count the sperm heads as
follows: 10 squares of the field.This number represents the concentration of
spermatozoa in millions per milliliter.

When counting a
15million/ml)all
sample 100 of
to that number,you
squares have to be
oligozoospermia
per
will milliliter.
counted.
(less get Adding
than the
concentration
"00000" of the
spermatozoa

is graded from A to D,

Grade A (fast progressive) sperms are those which swim forward fast
in a straight line - like guided missiles.
Grade B (slow progressive) sperms swim forward, but either in a curved or
crooked line, or slowly (slow linear or non linear motility).

Grade C (non-progressive) sperms move their tails, but do not move forward
(local motility only).

Grade D (immotile ) sperms do not move at all.

Immotile spermatozoa are to be counted in a defined number of squares.

Afterwards the motile spermatozoa are counted and classified into four
categories (from A "fast progressive" to D "no motility"). This procedure is to
be repeated in different areas of the grid to have the quality and motility to be
calculated on base of this data in percent.

Microscopic examination

Matility
Different quality of sperm).

categorle a.Rapid b.Slow

s of ·Non-progressive motility (NP):all other patterns of motility with an absence of progression,e.g:swimming

sperm in small circlesthe flagellar force hardly displacing the head,or when only a flagellar beat can be observed.

motilitv:
·Immotility (IM): no
·Progressive movement.
motllity (PR):
Spermatozoa If the motility is less
moving Ifthan 50%%
motility then vitalpentoxyphillene stimulation test is required
is Zero,then
actively,eithe
r linearly or test must be done.
ina large
cirele,regandl
ess of
speed.(Repre
sents the
 Microscopic examinalion

In Casc of Preance of Sem

Sample should be examined to determine the motility classification,aggregation.agglutination and the dilution
required for accurate assessment of sperm
concentration.
Asg
adherence either of immotile spermatozoa to each other.

·or of motile spermatozoa to mucus strands,non-sperm cells or debris is considered to be nonspecific

aggregation.
Agsfutination

Motile spermatozoa are aftached to each other Head-to-Head.Tail-to-Tail or in a mixed

way.(Immunological cause of infertility)

isolated<10 spermatoza per agglutinate,many free

Grade 1(+):
spermatozoa

moderate 10-50 spermatozoa per agglutinate,free

Grade 2(++):
spermatozoa
Grade 3(+++): large agglutinates of >50 spermatozoa,some spermatozna

Head-to-Head

Tall-to-Tall

Tall-tip-to-tall-tip

Mised

Tangle
Microscopic examination

fNoSprmmn:

First scan the whole slide and record if there are any motile and
immotile spermatozoa
If no sperm ,then add to semen specimen eqal volume of Ham's F10
media and centrifuge it at 1500 rpm for 10 minules.
Concentrate the sample in 250 micron and mix it well then Scan 10 micron at
x400 magnification.......econd presence of any sperm.
Second sample is useful.

·If azospermia: fructose level must be ordered to verify the integrity of

the vas and seminal vesicles

SPERM MORPHOLOGY
A drop of sample is placed in the sterile glass slide and covered using a cover
slip, name and SIDnumber should be mentioned. The sample is viewed under
light microscope with a magnification of 40x.

A Normal Sperm has:

A smooth, oval-shaped head that is 5-6 micrometers long and 2.5-

3.5micrometers wide (less than the size of a needle point)

A well-defined cap (acrosome) that covers 40% to 60% of the sperm head

No visible abnormality of neck, mid-piece, or tail

No fluid droplets in the sperm head that are bigger than one half of the sperm
head size.
The following categories of defects should be noted.

Head defects, namely large, small,

tapered,pyriform,globe,amorphous heads, vacuolated heads,
double heads and small acrosome.
Neck and mid-piece defects namely bent neck, abnormal mid-
piece (thin or thick).
Tail defects namely short, multiple,hairpin, broken,bent
tails,coiled tail.
Multiple defects
Agglutination
Pus cells/HPF
Debris
After completion of analysis the results should be noted in the
record and typed in the patient report entry column.
 Morphology assessment
Sperms must be stained for an accurate assessment

Strict criteria/Kruger/Tygerberg

The sperm head is oval,smooth-symmetrical outline, a length of 3-5 um and a width of

2-3um.

The head should have a well defined acrosome arca of 40-70% and vacuoles (=/<2)
that occupies~20% head area.

The mid-piece must be straight and slender, 0.5 um in width and 7-8um
long,straightly aligned to the head.

The tail must be straight and 45-50 um long.

To be classified as normal, the sperm must be normal in all portions (head,mid-

piece,tail).

At least 400 sperms must be scored on randomly chosen fields.

Normal Forms (%) = normal sperms/the total number of sperms evaluated x 100.

Diff-Quick Staining
Simple, quick staining method.

Moderate differentiation of structures--the acrosome is stained red and the

post acrosomal area dark red. Very thin smearsare necessary as the
background is influenced by the presence of protein in the seminal fluid and
tends to be dark.
The slides are fixed with fixative (Cytospray,Kinetik,Australia).
Diff Quik 1 for 5-6 seconds.
Clean water x 2. Removed excess moisture.
Diff Quik 2 for 10 seconds
2-3 dips in clean water.
It is left to dry by standing the slide on one end on absorbent paper towel.
Sperms stained by Diff-Quik are larger by comparison to H&E staining as the
cells did not undergo dehydration by alcohol.

Abnormal Sperms
Abnormal Sperms
1.Triple head sperm
2. Acrosome
reacted sperm
3. Sperm with no
acrosome
Sperm with a
tapering head
and swollen mid
-piece
 Morphology Assessment

Assessment is subjective due to the

wide variation in sperm sizes and
shapes.
% NF correlated well with the
fertilization rate in-vitro and
pregnancy rate [Kruger et al,
1996;Morgenthaler et al,1995]
Sperms with defective heads are more
likely to be immotile than sperms
without defects,and defective motile
sperms tend to show sluggish motility
as compared to normal sperms
[Aitken et al, 1995].

Vitality Assessment
1. Eosin-nigrosin (dead sperm stain pink/red)
2. Eosin (1%) (dead sperm stain pink/red)
3. Trypan (0.4%) blue (dead sperm stain blue)
4. Hypo-osmotic swelling test (HOS) (live sperm shows tail curling
Test 1,2 and 3 for diagnostic uses.
Usually 1:1 ratio of semen to dye mixture, mix well and smear
onto a slide. Read immediately at x40 objective, count 200sperms
Test 4 is use to choose live (immotile) sperm for ICSI
Dead sperms will not react in HOS while live sperm will take up fluids
causing their tails to curl within 5 min and stabilize at 30 min.
Therefore viable sperms may be selected for ICSI [Lin et al,
1998;Cayan et al, 2001].
 Vitality Assessment

Dead
sperms
are
stained
pink/re Live sperms shows curling tails
d
Thank you for your attention