SEMEN ANALYSIS : TEST FOR MALE FERTILITY

 

IMMUNOLOGY AND CELL BIOLOGY DEPARTMENT

ARKAD MEDLAB SOLUTIONS


 STANDARD OPERATING PROCEDURE FOR SEMEN ANALYSIS

OBJECTIVES
• To evaluate male fertility in an infertile marriage.


• To substantiate the effectiveness of vasectomy


• To detect semen on the body or cloth of a suspected rape victim at the crime scene.


• To identify blood group substances to exonerate or incriminate a criminal suspect.


• To rule out paternity on grounds of complete sterility.


PATIENTS’ PREPARATION
• Explain the procedure to the patient
• Instruct the patients to abstain from sexual activity for 2 to 3 days before collecting the specimen.
• Give the patient the proper container for the sperm collection.
• Instruct the patient to avoid alcoholic beverages for several days before the collection.

SAMPLE COLLECTION AND DELIVERY
• Ideally the sample should be collected in the privacy of a room near the laboratory. If not, it should be delivered to the laboratory within 1 hour after collection.

• The sample should be collected after a minimum of 48 hours and no longer than 7days of sexual abstinence. The name of the man, period of abstainence, date and time of collection should be recorded. When the duration of abstinence is more than 7 days, sperm motility, and concentration may be decline; if the duration of abstinence is 48hrs, sperm concentration may be reduced.

• The sample should be obtained by masturbation and ejaculated into a clean, wide –mouthed glass or plastic container. The container should be warm to minimize the risk of cold shock. In cases in which masturbation is not possible or against individual’s values, the specimen can be collected in a non-spermicidal condom. Coitus interruptus is not acceptable as a means of collection because it is possible that the first portion of the ejaculate, which contains the highest concentration of spermatozoa, will be lost.


LABORATORY PROCEDURE

MACROSCOPIC EVALUATION

• Appearance

o A normal sample has a grey –opalescent, is homogenous and liquefies within 60 mins at room temperature. The  sample may appear clear if the sperm concentration is too low or brown when red blood cells are present(haematosperia).

• Consistency (viscosity)

o It can be estimated by gentle aspiration into a 5ml pipette and then allowing the semen to drop by gravity and observing the length of the thread formed.

• Volume

o The volume  of the ejaculate should be measured either with a graduated cylinder or by aspirating the whole sample into a wide - mouth pipette.

• pH

o The pH is determined by acidic secretions of the prostate and alkaline secretion of the seminal vesicles. It should normally be in the range of 7.2-8.0.

     MICROSCOPIC INVESTIGATION
• MOTILITY

o A drop of the semen is delivered onto a clean glass slide and covered with 22x22mm coverslip. The microscopic field is scanned systemically with  X40 objective lens and a total of  100 spermatozoa are counted. The motility of the sperms is graded in percentage  whether it shows

a) active linear forward progressive motility
b) slow or sluggish progressive motility
c) non progressive motility
d) immotility
 



• Agglutination

o Agglutination of sperms means that motile spermatozoa stick to each other, head to head, middle to middle, tail to tail, or mixed. In case of agglutination, semen culture must be performed in order to exclude infection with Escherichia coli.

• Sperm Viability

Vital staining of the spermatozoa allows quantification of the fraction of living cells independently of their motility. Live and dead spermatozoa are distinguished by adding one drop of 0.5% eosin y stain to one drop of semen at room temperature and smear the mixture on microscopic slide. Using the X40 objective, 100 spermatozoa are classified as either colored orange-red, if the stain has passed through the membrane and therefore the cell is dead, or non-stained, the cell being considered alive.

• Counting the spermatozoa

In the haemocytometer method, a 1:20 dilution from each well mixed sample is prepared by diluting 50ul of liquefied semen with 950ul diluent. The latter is prepared by adding 50g of sodium carbonate, 10ml of 35%(v/v) formalin and make up the solution to a final volume of 1000ml.

Using a Pasteur pipette, charge an improved Neubauer ruled chamber with well mixed solution. Wait 3-5minutes for the spermatozoa to settle. Using the X10 objective, count the number of the sperm cells in an area of 2 sq mm. (2 large squares). Calculate the number of spermatozoa in 1ml of fluid by the number counted by 100 000.

• ANALYSIS OF THE MORPHOLOGICAL CHARACTERISTICS OF SPERMATOZOA

i. Make a thin smear of the liquefied well-mixed semen on a slide. While wet, fix the smear with 95% v/v ethanol for 5-10 minutes, and allow to air-dry.

ii. Wash the smear with sodium bicarbonate-formalin solution to remove any mucus which may be present. Rinse the smear with several changes of water

iii. Cover the smear with dilute (1 in 20) carbon fuchsin and allow to stain for 3 minutes. Wash off the stain with water

iv. Counter stain, by covering the smear with dilute (1 in 20) Loeffler’s methylene blue for 2 minutes. Wash off the stain with water. Drain, and allow the smear to air-dry

Staining results

Nucleus of head………………………………………Dark blue
Cytoplasm of head……………………………………Pale blue
Middle piece and tail………………………………….Pink-red

NOMENCLATURE FOR SEMEN VARIABLES

Normospermia
Normal sperm count, motility and morphology


Oligozoospermia
Sperm count fewer than 20 million/ml.


Asthenozoospermia
Fewer than 50% spermatozoa with forward progressive movement.


Teratozoospermia
Fewer than 50% spermatozoa with normal morphology.


Oligoasthenoteratozoospermia
Sperm count, forward progressive movement and morphology are disturbed (< 50%) .


Azoospermia
No spermatozoa in an ejaculate
Aspermia
No ejaculate.

COMPILED BY MEDICAL LAB. SCIENTIST, ANTHONY D. DZAMESI.

To order for your semen analysis test and other fertility test for females contact Arkad medlab solutions: 

arkadmedlabsolutions@gmail.com

Also contact us for quality herbal drug to cure male infertility.

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