SPERMATOZOA I :
Sample collection
The sample should be collected after a minimum of 48 hours but not longer than seven days of sexual abslinence. To reduce the variability of semen analysis results, the number of days of sexual abstinence should be as constant as possible

Definitons
Normal ejaculate as defined by the reference values
Oligozoospermia : Sperm concentration less than 20.000.000 permatozoa/ml or less than 40.000.000 spermatozoa per ejaculate
Asthenozoospermia : Less than the reference value for motility
Teratozoospermia : Less than reference value for morphology
Oligo-astheno-teratozoospermia : Signifies disturbance of all three variables (combinations of only two prefixes may also be used)
Azoospermia : No spermatozoa in the ejaculate
Aspermia : No ejaculate

Reference values

Nomal values appromimatively may occur from 1 to 4 times for
sperm concentration and 1 to 2 times for sperm motility.

ROUTINE ANALYSIS
The fertilizing potential of sperm depends on number of spermatozoa, their morphology (shape) their motility, and their ability to transfer a perfect genetic material (DNA) to the egg at the time of fertilization.
It is well known that 40-50% of all infertility problems are due to sperm abnormalities arising during the production of sperm or due to functional problems or in combination with other medical or toxic factors.

Without macroscopic examination (Viscosity and liquefaction time, appearence, volume) the semen sample is analyzed for its concentration in sperm, its percentage of actively moving (motility and velocity) as well as normal shaped sperm (morphology).

Sperm concentration can be performed with a Neubauer haematocytometer after dilution or directly evaluated in a Makler chamber :

Reference values :
Volume : 2.0 ml or more
pH : 7.2 or more
Sperm concentration : > 20.000.000 spermatozoa/ml
Total sperm number : > 40.000.000 spermatozoa /ejac.
White blood cells : < 1000 / ml

Sperm motility
A simple grading system is recommended which provides an assessment of sperm motility without the need for complex equiprnent.The motility of each spermatozoon is graded 'a', 'h', 'c', or 'd' :
a= rapid progressive motility
b= slow or sluggish progressive motility
c= nonprogressive motility
d= immotility.
Other methods of assessment of sperm motility, including cornputer-aided sperrm analysis (CASA), can be used too.

Reference values :
Volume : 2.0 ml or more
pH : 7.2 or more
Sperm concentration : > 20.000.000 spermatozoa/ml
Total sperm number : > 40.000.000 spermatozoa /ejac.
Whitc blood cells : < 1000 / ml

Sperm vitality test

This should be determined if the percentage of
immotile spermatozoa exceeds 50%.
1 Eosine-nigrosine test : The proportion of live spermatozoa can be determined by using staining techniques that are based on the principle that dead cells with a damaged plasma membrane take up certain stains
2 HOS test - Hypo Osmotic Swelling Test
Simple tcst based on the semipermeability of the intact cell membrane when an influx of water results in an expansion of cell volume.

1 - Eosine-nigrosine Test - 2 - Hypo Osmotic Swelling Test

Normal values : Vitality : > 75% live

Sperm morphology :
Staining methods :
The Papanicolaou stain and The Shorr stain are the method most widely used

A rapid staining method such as the Diff-Quik is used in routine too. But Some smears stained by rapid procedures may have background staining and may not always give the same quality as the Papanicolaou stain or Shorr stain.

Photos and schematic drawings of some abnormal forms of human spermatozoa.



Reference values : Morphology : > 30 % with normal forms

Another cells :

Leukocytes
When the number of leukocytes in semen is high, rriicrobiological tests should be performed to investigate if there is an accessory gland infection
Round cells
Immature germ cells 'l'he round cells other than Icul<ocytes include round spermatids, spermatocytes, spermatogonia, and exfoliated epithelia1 cells.
These are often degenerating and difficult to identify.

1 -  spermatogonia, 2 -  spermatocyte I
3,4,5 – spermatids , 6 – spermatocyte II

The different types of immature germ cells appearing in semen are usually indicative of disorders of spermatogenesis.

Antibody-coating of spermatozoa

The presence of agglutination is suggestive of, but not sufficient evidence to prove the existence of an immunological factor of fertility.
The screening test for antibodies is performed on the fresh semen sample and makes use of either the MAR Test ( mixed antiglobulin reaction test, using IgG coated red blood cells) or the Immunobead method (IgA, IgG, or IgM coated latex) .
Sperm antibodies in semen belong to the immunoglobulin classes IgG, IgA or rarely IgM. There are some data suggesting that IgA antibodies may have greater clinical importance than IgG antibodies.
The presence of sperm antibodies coating the spermatozoa is typical of and is considered to be specific for immunologic infertility.

sperm antibodies have been shown to impair fertility and may account for up to 8 % of couples whose infertility is unexplained.
Normal values :
Immunobeads Test : >20 % sp.with adherent latex particules
MAR Test : 10 % sp. with adherent sensibilized particules
when 50% or more of the motile spermatozoa have particles adherent, The diagnosis of immunologic infertility is probable and must be confirmed by sperm-mucus interaction tests.

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Semen culture
While contamination by microorganisms is not synonymous with clinical infection, their presence can have a dramatic influence in culture media during human assisted conception process.
Therefore, a bacteriologic control of semen is necessary before each ART process.