TO ANSWER THE QUERIES-----
What is the problem??
Where is the problem???
MALE REPRODUCTIVE ORGAN
TESTIS IS FACTORY, PRODUCT IS SPERM
M ALE INFERTILITY
IF PRODUCT (SPERM) IS NOT COMING TO THE
MARKET (SEMEN) OR OF POOR QUALITY:
REASONS MIGHT BE:
1.MALFUNCTIONING OF MANAGER
(HYPOTHALAMO PITUITARY) :PRETESTICULAR
2.MALFUNCTIONING OF FACTORY (TESTIS) :
3. MALFUNCTIONING IN SUPPLY LINE (Obstruction
in epi/Vas) : POST TESTICULAR
SO PROBLEM MAY BE
BESIDES SEMEN ANALYSIS :
We can advice to Go for andrological evaluation
Which includes- FSH
testicular volume by usg
FSH IS LOW ( < 4 mIU/ml ) : Means adequate
messangers is not released from hypothalamus(
Hypogonadotrophic Hypogonadism )
TESTICULAR VOLUME WILL BE LOW
TREATMENT IS POSSIBLE BY INJECTING
FSH IS RAISED (normal :4-14 mIU/ml): twice than
normal : Means testis is not functioning well hence
(Hypothal -Pituit) sends large amount of
Concomitantly the volume of the Testis will be Low
(normal >12 cc)
DIAGNOSIS : PRIMARY TESTICULAR FAILURE
TREATMENT NOT POSSIBLE ( ? )
FSH IS IN NORMAL RANGE (4-14 )
TESTICULAR VOLUME IS NORMAL (> 12 CC )
FRUCTOSE IS ABSENT IN EJACULATE
DIAGNOSIS : OBSTRUCTION IN OUTFLOW
most common at vaso-epididymal junction
TREATMENT : SURGICAL BY-PASS OF
OBSTRUCTION OR RETRIEVAL OF SPERMS
FROM TESTIS AND DOING icsi
PARTS OF SEMINAL FLUID
Source of secretions Volume Characteristics
0.1-0.2 CC SPERM PRESENT
Urethral and bulbourethral glands 0.1-0.2cc Viscous, clear
Prostate 0.5-1.0cc Acidic, watery
Seminal vesicles 1.0-3.0cc Gelatinous, fructose positive
Complete ejaculate 2.0-5.0cc Liquefies in 20m-25m
Semen analysis should begin with a simple
inspection soon after liquefaction.
30 minutes, to 1 hour after ejaculation, to prevent
dehydration or changes in temperature from affecting semen quality.
making semen evaluation difficult.
In these cases, additional treatment, mechanical mixing or
enzymatic digestion may be necessary.
1. Some samples can be induced to liquefy by the addition of an equal
`volume of physiological medium (e.g. Dulbecco’s phosphate-buffered
saline; followed by repeated pipetting.
2. Inhomogeneity can be reduced by repeated (6–10 times) gentle
through a blunt gauge 18 (internal diameter 0.84 mm) or gauge 19 (internal
diameter 0.69 mm) needle attached to a syringe.
3. Digestion by bromelain, proteolytic enzyme may help to promote
Comment: These treatments
may affect seminal plasma
and sperm morphology,
and their use must be recorded.
Dysfunction of seminal vesicle
Or prostate (fibrinogen from prostatic fluid degrades
fibrin in coagulum)
The volume is best measured
by weighing the sample in the
vessel in which it is collected.
Collect the sample in a pre-
weighed, clean, disposable
Weigh the vessel with semen
Subtract the weight of the
Calculate the volume from the
sample weight, assuming the
density of semen to be 1 g/ml
the volume can be measured directly.
Collect the sample directly into a
modified graduated glass measuring
with a wide mouth. These can be
Read the volume directly from the
graduations (0.1 ml accuracy).
•Comment 1: Low semen volume is characteristic of
obstruction of the ejaculatory duct or congenital bilateral
absence of the vas deferens (CBAVD) condition in which the
seminal vesicles are also poorly developed.
•Comment 2: Low semen volume can also be the
result of collection problems (loss of a fraction of the
ejaculate), partial retrograde ejaculation or androgen deficiency.
•Comment 3: High semen volume may reflect active
exudation in cases of active inflammation of the accessory
WHY IS IT IMPORTANT
Note: Measuring volume by
aspirating the sample from the
specimen container into
a pipette or syringe, or decanting it into a
measuring cylinder, is not
because not all the sample will be
retrieved and the volume will therefore
The volume lost can be between 0.3 and
1. After liquefaction, the viscosity of the sample can be estimated by gently
aspirating it into a wide-bore (approximately 1.5 mm diameter) plastic
disposable pipette,allowing the semen to drop by gravity and observing
the length of any thread.
2. A normal sample leaves the pipette in small discrete drops.
3. If viscosity is abnormal,the drop will form a thread more than 2 cm long.
4. The viscosity should be recorded as abnormal when the thread exceeds
5. In contrast to a partially unliquefied sample, a viscous semen specimen
exhibits homogeneous stickiness and its consistency will not change
with time. High viscosity can be recognized by the elastic properties of
the sample, which adheres strongly to itself when attempts are made to
6. The methods to reduce viscosity are the same as those for delayed
Comment: High viscosity can
interfere with determination of
sperm motility, sperm
concentration, detection of
and measurement of
The pH of semen reflects the balance between the
pH values of the different accessory gland
the alkaline seminal vesicular secretion and
the acidic prostatic secretion.
The pH should be measured after liquefaction at a
uniform time, preferably after 30 minutes, but in any
case within 1 hour of ejaculation
since it is influenced by the loss of CO2 that occurs
In azoospermic male:
If the pH is <7.0 in a semen sample with low
volume , there may be ejaculatory duct
obstruction or congenital bilateral
absence of the vas deferens
pH >7.8--indicates dysfunctional
: Semen pH increases with time, as natural
buffering decreases, so
high pH values may provide little clinically useful
Depth of wet preparations=VOL. OF SAMPLE X AREA OF SPREAD
USUALLY WE NEED A DEPTH OF 20 um FOR ASSESSMENT
chamber depth of less than 20 um constrains the rotational movement
spermatozoa (Le Lannou et al., 1992; Kraemer et al., 1998).
If the chamber is too deep, it will be difficult to assess spermatozoa
as they move in and out of focus
AREA OF COVER SLIP VOL. DEPTH
22mm X 22mm 10 ul 20.7 um
21mm X 26mm 11ul 20.1 um
18mm X 18mm 6.5ul 20.1 um
WET PREPARATION ON SLIDE
The volume of semen and the
dimensions of the cover slip must be
At a glance we get idea about:
any other cells besides sperm
NUMBER OF SPERMATOZOA PER HPF IN WET
estimate the number of spermatozoa per HPF
(×200 or ×400).
One HPF is equivalent to approximately 16 nl (at ×200) or
4nl (at ×400)
If spermatozoa are observed, count them, determine the
If there are 100 spermatozoa per
high-power field (HPF) of 4 nl in the
initial wet preparation, there are
theoretically 25 per nl (25 000 per
ul or 25 millions/ ml).
NON-SPECIFIC AGGREGATION OF
SPERMATOZOA IN SEMEN
Views of spermatozoa aggregated with an epithelial
cell (a), debris (b) or spermatozoa (c, d).
DEGREE OF AGGLUTINATION
B. Tail-to-tail (heads
are seen to be free and
move clear of
D. Mixed (clear headto-
head and tail-to-tail
E. Tangle (heads and
tails enmeshed. Heads
are not clear of
as they are in tailto-
Progressive motility (PR): spermatozoa moving actively,
either linearly or in a large circle, regardless of speed.
Non-progressive motility (NP): all other patterns of
motility with an absence of progression, e.g. swimming in small circles,
the flagellar force hardly displacing the head, or when only a flagellar
beat can be observed.
Immotile (IM): no movement
MOTILITY REPORTED AS
Initially scan the SLIDE for
Pro. Motile cells count Nonpro.
motile spermatozoa IMmotile
With experience, it may be possible to
score all three categories of sperm
movement at one time, and to score
larger areas of the grid.
Lower reference limit
The lower reference limit for TOTAL MOTILITY (PR +
NP) is 40% (5th centile,
The lower reference limit for PROGRESSIVE MOTILITY
(PR) is 32% (5th centile,
Comment: The total number of progressively motile
spermatozoa in the ejaculate
is of biological significance. This is obtained by
multiplying the total number of
spermatozoa in the ejaculate by the percentage of
progressively motile cells.
In this case –checked for any infection,agglutination
In this case
Gave antibiotic t/t prophylactically
Did HOS test –then ICSI was done in 8 oocytes
With HOS positive Sperms
BY NEUBAUER CHAMBER(RECOM. BY WHO)
OR MAKLER’S CHAMBER,CASA-TOO EXPENSIVE
AND HAVE LITTLE ADVANTAGE OVER STANDARD
THE IMPROVED NEUBAUER HAEMOCYTOMETER
The improved Neubauer haemocytometer has two
separate counting chambers,
each of which has a microscopic 3 mm × 3 mm
pattern of gridlines etched on the glass surface.
It is used with a special thick coverslip (thickness
number 4, 0.44 mm), which lies over the grids and
is supported by glass pillars 0.1 mm above the
Each counting area is divided into nine 1 mm × 1
These grids are referred to by the numbers
1 2 3
4 5 6
7 8 9
Sperm counted in no 5 grid
5 squares to 25 squares
With a depth of 100 um, each grid holds 100 nl.
Four of these grids (nos 1, 3, 7 and 9) contain
four rows of four squares, each holding 6.25 nl;
two grids (nos 2 and 8) contain four rows of five
squares, each of 5 nl;
two grids (nos 4 and 6) contain five rows of four
squares, each of 5 nl; and
the central grid (number 5) contains five rows
of five squares, each of 4nl.
(correspond to wet preparation)
Each of the 25 squares of the central grid (number
5) is subdivided into 16 smaller squares
1 2 3
4 5 6
7 8 9
Thus, grids 1, 2, 3, 7, 8 and 9 each have
four rows holding 25 nl per row,
while grids 4, 5 and 6 each
have five rows holding 20
nl per row.
Depending on the dilution and the number
of spermatozoa counted, different areas of
the chamber are used for determining
For 1 + 19 (1:20) and 1 + 4 (1:5)
dilutions, rows from grid number 5 are
assessed and, when necessary, from
grids numbers 4 and 6
For 1 + 1 (1:2) dilutions,all nine grids can
be assessed if necessary to achieve a
count of 200 spermatozoa
1 2 3
4 5 6
7 8 9
As the central grid (number 5) of the
improved Neubauer chamber holds
100 nl, there would be 2500 spermatozoa
Diluting the sample 1 + 4 (1:5) would
reduce the background and the sperm
number to about 500 per grid, which is
sufficient for an acceptably low sampling
If there are 10 spermatozoa per HPF of the
wet preparation, there would be 2.5 per nl
and 250 per central grid. Diluting the
sample 1 + 1 (1:2) as suggested would
reduce the background and the sperm
number to about 125 per grid; this would
375 in the three grids numbered 4, 5 and
6—again, this is suffi cient for an
low sampling error.
COUNT only intact spermatozoa (having head
Do not count motile pinheads
Do not count immature germ (round) cells.
Do not assess overlapping spermatozoa and
those lying with the head on edge; these cannot
be analysed adequately. They should not be
present in a good smear , but may occur when
debris and a large amount of particulate material
are present (such as in viscous sample.
These samples should be washed and the slides
prepared before staining.
COUNT :WHAT I DO
Assess the sperms concentration from slide
And make a dilution of 1:20 sperm according to the
chart given below.
Charge the Neubauer chamber
Count the squares in the central grid usually 5
X –multiply by 106 per ml.
Make a dilution and count as follows:
e.g. if 6 spematozoa are counted in 25 squares with semen diluted 9 X(1+9)
Then count =6/10 x 106 per ml or 600000 sperms per ml
Ref: ICMR AND WHO COLLABORATING CENTRE FOR RESEARCH IN
Dilution factor Correction factor for no. of squares counted
the sperm concentration X volume of the whole
ejaculate== sperm number
The lower reference limit for sperm concentration is 15 × 106/ ml
The lower reference limit for total sperm number is 39 × 106
spermatozoa /ejaculate (5th centile, 95% CI 33–46 × 106).
Normozoospermia Normal ejaculate
Oligozoospermia Sperm concentration fewer than 15x106/ml
Asthenozoospermia <40% spermatozoa with total motility(pro. And non- prog) or <32%
spermatozoa with pro. movement
Teratozoospermia Fewer than 30% spermatozoa with normal morphology
Signifies disturbance of all three variables (combination of only two
prefixes can be used)
Azoospermia No spermatozoa in the ejaculate
Aspermia No ejaculate
Certain terms used in reporting
WHY REPORTING 100% GLOBOZOOSPERMIA
Even if we do icsi with globo. Sperm
There is fertilization failure –why?
Sperm vitality, is estimated by assessing the membrane integrity of the
If <10% progressively motile spermatozoa----do vitality assessment
The percentage of live spermatozoa is assessed by identifying those with
an intact cell membrane, from dye exclusion or by hypotonic swelling.
The dye exclusion method damaged plasma
membranes, such as those found in non-vital (dead) cells, allow entry of
The hypo-osmotic swelling test presumes that only cells
with intact membranes (live cells) will swell in hypotonic solutions.
This is useful when staining
of spermatozoa must be
avoided, e.g. when choosing
spermatozoa for ICSI.
Spermatozoa with intact
membranes swell within 5
minutes in hypo-osmotic
medium as indicated by
curling of the tail
While dead sperms shows no
all flagellar shapes are
stabilized by 30 minutes
30 minutes incubation for routine diagnostics; but
5 minutes incubation when spermatozoa are to be processed for
Preparing the reagents
1. Swelling solution for diagnostic purposes: dissolve 0.735 g of
sodium citrate dihydrate and 1.351 g of D-fructose in 100 ml of
purified water. Freeze 1-ml aliquots of this solution at –20 °C.
2. For therapeutic use: dilute the medium to be used 1 + 1 (1:2) with
sterile, purified water.
8. Tally the number of unswollen (dead) and swollen (vital) cells with
the aid of a laboratory counter.
9. Evaluate 200 spermatozoa in each replicate, in order to achieve an
acceptably low sampling error
VOLUME 1.5 ml (1.4-1.7)
39 million (33-46)
Count/ml 15 million/ml(12-16)
Total MOTILITY 40 %(38-42)
% NORMAL FORMS 4 % (3.0-4.0)
SOME LOWER REF. VALUE
Pro. Motility % 32% (31-34)
Vitality 58% (55-63)
WBC( peroxidase positive) <1.0 million/ml
KRUGER’S CRITERIA FOR SPERM
For this test, freshly ejaculated sperm are smeared on a
slide and stained using a morphology staining product
for human sperm. Sperm are judged as normal based
on the following criteria:
Head must be oval in shape with smooth contours, 5-6
µm in length and 2.5 to 3.5 µm wide with the acrosome
taking up 40-70% of the head.
Neck and mid-piece must have no abnormalities and a
cytoplasmic droplet (a remnant from sperm production)
if present must not be larger than half the size of the
Tail must not be coiled or bent and should not have a
droplet at the end.
After 200 individual sperm are counted at a
magnification of 1,000 times, the percent normal
forms is calculated. The prognosis is based on the
>=15% normal:Normal range - Good prognosis
5-14% normal:Sub optimal range - Prognosis is fair
to good, however, the lower the percent normal, the
lower the chance of successful fertilization
0-4% normal:Poor prognosis - Will usually need
IVF with intracytoplasmic sperm injection (ICSI)