What we the non-ART specialists need
to know about routine Semen examination??:
General overview:
Part
1: Instruction to the male partner :-- The chamber
Asst must explain in local language abut details of semen collection
particularly days of abstinence, whole sample collection, not to use any
lubricants and special semen collection tube to be collected from lab. Two days
abstinence is desirable but not exceeding 5 days .That can falsely increase dead immotile sperm & pus cells
too
Why sperm density is so relevant ?? Ans:-The probability of conception
increases with increasing sperm
concentration upto approximately 40-50
million / ml but does not rise
further with higher sperm densities .
What
is meant by Physical & Chemical examination of
semen Ans: Physical examination is often ignored by Lab
technician which is imprecise,. In physical examination one has to check for 6
characteristic like 1)Volume 2) pH 3) Liquefaction time 4) Appearance(colour) 5) Smell
6 ) opacity . Traditional Chemical examination usually
comprises of fructose estimation & routine microscopy includes: 1) sperm concentrations
(density) & Total sperm count 2)
morphology 3) Motility Total
motility, : & Progressive motility 4) Agglutination 5) pus cells 6) vitality(alive) 7) RBC .
What
are Special tests :: Some special tets are done at higher centers : CASA, Kruger tets for morphology, Quantum of
ROS, Sperm DNA concentration , Immunobead tets, MAR test, are rarely performed and don’t come under the
preview of routine analysis.
How
to calculate sperm density : By what slide?? Sperm concentration is calculated as number
of sperms per ml. It is classified into
1) azoospermia (no sperm cells even in centrifuges specimen) 2) mild < 15 million /ml of liquefied semen 3) moderate
5-10 million /ml of liquefied semen 4) severe oligospermia .< 5 million million /ml of liquefied semen.
Word of Caution on routine semen anlyis –if any single
parameter defect :- Such an report should only be disclosed
to the concerned couple after three
consecutive examinations done at
different laboratories at an interval of one month. It will be better if
he can avoid some the / all correctable factors which can decrease or modify
his sperm density. Such modifiable factors are A) tight under wares, 2) control
of MAGI(Male Accessory Gland Infections), control of STI, C) Smoking and
substance use D) Semen Collection errors
(not to mix saliva or add tap water after ejaculation if some semen spills at
floor of collection room which will be described in details below and E)
correction of surgical pathology like Hydrocele , retractile testis.
Q. What other tets if repeated poor seminal parameters?? Serum
endocrine parameters including FSH, LH , Free T4 may be asked and corrected if
second examination too is suboptimal .including karyotype. Endocrine and genetic evaluation is indicated in men
with severe oligospermia
To enquire any childhood trauma
or viral diseases . Other Lab
reports:-Virology, Urine culture, P/R examination-for possible paplable seminal vesicles and enlarged prostate??
Classificationof
sperm density density after three conseceutive tets??
Azoospermia
---- No sperms seen
Extreme
oligospermia -- < 1x 106 /
ml
Severe oligospermia – 1-5 x 106 /ml
Moderate
oligospermia 5-10 x 106 /ml
Mild
oligospermia 10-20 x 108 / ml
Normal ---
>20x 106/ml
What do we
men by Total sperm count?? Total sperm count is the product obtained by
multiplying semen volume and sperm concentration
Note : The total
count can be really normal in some oligozoospermic men when
volume is high(increased Prostatic activity & or urethral glands) .
Why we are worried about sperm count(sperm density) ?? Azoospermia
How to assessment of
sperm Concentration
The semen is mixed thoroughly.
Concentration can be determined by three
methods:
· Hemocytometer method
· Makler’s chamber
· Micro cell.
· The dilution of the sample
depends on initial wet smear examination
. If no spermatozoa are found the whole
sample is centrifuged and pellet
is reexamined.
Sperm per field
of vision <15 – Then dilution – 1+4 (1:5) , Semen – 100, Diluent –
400
15-40 –Dilution
– 1+9 (1:10) , Then Semen – 50, Diluent – 450
40-200 ,
dilution – 1+19 ( 1: 20 ) , Then Semen – 50 , Diluent – 950 ,
Ø 200 , dilution – 1+ 49 ( 1 : 50 ) , Then
Semen – 50 , diluents – 2450
A)
Neubauer’s Hemocytometer (most ordinary lab uses this
method)
10 ul (micro liter ) of diluted specimen is transferred by touching
the edge of the cover glass on
Neubauer’s hemocytometer. Phase contrast microscope is used at a magnification of 40 X .
Only normal spermatozoa are included in
the count. If sample contains less than 10 sperms per large squares are
assessed. If sample has > 40 spermatozoa per large square 5 large squares are assessed . The conversion factor is used to determine final
concentration is semen .
Disadvantage ;
Large dilutions have an effect on precision
B)
Makler
chamber :-Cost
about such special slide is about Rs 30-35,000/-mostly used in higher centers, like andrology lab, ART
centers ,those who caters IUI and at least 3-5 dozens of top d quality Lab of repute per metro city of India .
No dilution is required.
Liquefied and stirred raw semen is put
on the chamber covered with a cover slip
an seen under 20 X magnification .
3) CASA
Although CASA system can be used to automatically measure sperm concentration errors
are encountered when there
are high and low concentrations
significant agglutinates or large amount of significant
debris. The manual count can be obtained
with a coefficient of variation of less than 10 %. The CASA instrument
digitalizes the electrical
signals that result from repeated video
scans of a field of sperm The
sperm cell is recognized because of
its optical contrast
with the background. CASA
provides numbers that quantitative the vigor and pattern of movement on a per
sperm basis.
When no motile sperms are observed a sperm viability test should be done to differentiate viable non motile sperms from dead sperms
as it is important to identify living
non motile sperms for ICSI
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