Sperm Analysis
Human semen consists of spermatozoa suspended in plasma-like fluid and is formed at ejaculation. Only the spermatozoa and a small number of secretions are produced in the testis and the epididymis (5% of total volume). The bulk of the semen consists of the secretions of seminal vesicles (46-80%) and the prostate (13-33%). Bulbourethral and urethral glands contribute about 2-5% of the total volume.
These secretions not only affect the concentration but also the function of the spermatozoa. During intercourse, each component is expelled in the posterior urethra by the process of emission followed by ejaculation out of the urethra. The mixing of the components takes place after ejaculation.
The ability to penetrate the eggs is also acquired in the epididymis. Seminal vesicles provide an important energy source in the form of fructose that enhances the motility of spermatozoa. Prostaglandins and fibrinogen-like substances are also added to seminal fluid by seminal vesicles. Prostatic fluid provides a number of enzymes, spermine (a bacteriostatic substance), citrates, calcium, and zinc. The bulbourethral and urethral glands contribute mucoproteins and IgA to semen. Thus, a disease of any of these parts of the male genital tract may have a profound effect on both the quality and the quantity of semen and may lead to infertility.
INDICATIONS FOR SEMEN ANALYSIS
The Human semen include infertility, hypogonadism, follow up after a vasectomy, prior to donations for artificial insemination and storage of semen before radiotherapy, etc.
SAMPLE COLLECTION OF SEMEN
A period of abstinence is important
as it affects both the quantity and motility of spermatozoa. Ordinarily, abstinence of 3-5 days is adequate. It is more convenient and practical to produce the specimen in the laboratory. However, some patients may not feel relaxed and comfortable in the laboratory atmosphere and stress is known to affect both the quality and the quantity of semen. In such cases, it might be more fruitful to ask the patient to produce the specimen at home and quickly transport it to the laboratory.
as it affects both the quantity and motility of spermatozoa. Ordinarily, abstinence of 3-5 days is adequate. It is more convenient and practical to produce the specimen in the laboratory. However, some patients may not feel relaxed and comfortable in the laboratory atmosphere and stress is known to affect both the quality and the quantity of semen. In such cases, it might be more fruitful to ask the patient to produce the specimen at home and quickly transport it to the laboratory.
The Human semen specimen should be collected in the morning to allow sufficient time for its analysis. Masturbation is the ideal method for producing the semen specimen. However, due to psychological or religious reasons, this might not be possible in some patients. In such instances, coitus interruptus can be resorted to but a part of the ejaculate may be lost by this method. It is important that both the pathologist and the patient be aware of this fact. Condoms must never be used for the collection of semen by intercourse. A clean, dry, wide-mouth glass or plastic jar should be used as the semen container. Its lid must not be rubber-lined. Detergents, water, and rubber are injurious to sperms. Specimens should be transported to the laboratory at a temperature as close to 37°C as possible and be delivered to the laboratory in less than 1 hour.
PHYSICAL EXAMINATION OF HUMAN SEMEN
Transfer the semen into a scrupulously clean graduated small cylinder. Note the volume, color, appearance and the pH. Normally, the human semen, soon after ejaculation, forms a gel-like clot that liquefies in 5-20 min. and therefore, by the time it is brought on the workbench, it has usually liquefied. If not, it should be liquefied before analysis by adding 510 drops of 0.2% Į-amylase. The absence of liquefaction in a semen sample must be noted.
The viscosity of semen should be assessed. It can be measured by dropping a drop of semen from a 10 cm-long capillary tube containing 0.1 ml semen. The time taken by the drop to form and leave the capillary tube is a measure of its viscosity.
SPERM COUNTING
Visual assessment
Place a drop of Human semen on a clean glass slide and lightly place a coverslip over it. Examine the slide under the high-power objective of a microscope to make a visual assessment of the sperm count and to determine the need for any dilution.
Dilution
The diluent used is 3.5% buffered formal saline prepared by dissolving 5 g sodium bicarbonate, 1 ml of 35% formalin and distilled water to make a total volume up to 100 ml. Five ml of the saturated aqueous solution of gentian violet can be added to this fluid to stain the sperms. The fluid immobilizes the spermatozoa and facilitates counting. Normally 1 in 20 dilutions are made by adding 50 µl of well-mixed and liquefied semen to 950 µl of diluent (Sahli pipette). However, 1 in 10 dilutions is recommended and 1 in 50 dilutions may be required if the sperm count appears to be high.
Counting Procedures
Improved Neubauer chamber (Haemocytometer) is used for counting. Both the chamber and the coverslip must be washed with distilled water and dried before use. The coverslip is then pressed on the central area until all the air is out and birefringent rings appear on the side. The diluted Human semen is carefully mixed and the chamber is charged using a Pasteur pipette. The chamber is then examined by using x10 objective of the microscope. Sperm are counted in the four large corners and one large central square (WBC counting area). It is important that loose tails and germinal cells are not counted. At least 200 spermatozoa must be counted. If these are not available in these 5 squares, more squares must be counted.
ASSESSMENT OF SPERM MOTILITY
Assessment of motility must be performed soon after the production of the sample, 3 and 6 hours later. It is important to remember that sperm require at least 10 ȝm of depth for free movement. A drop of well-mixed undiluted Human semen is placed on a warm clean slide and very lightly covered with a coverslip. The slide is allowed to rest on the microscope stage or bench until µstreaming of the semen stops and is then viewed under the microscope. Both motile and immotile sperm are counted at least 5 fields with a minimum count of 200. The count should be performed in duplicate and the average recorded. Only forward movement of the sperms is taken as positive. Percentage motility is then calculated. The sperm count can be calculated using the formula:
Motilespermcount Spermcount/ml % motility semen vol
More objective results can be obtained by the following procedure:
1. About 30 min after collection transfer the Human semen in a capped tube. Gently mix by inverting the tube several times.
2. Pipette one drop of semen onto a clean glass slide; place a clean coverslip over it.
3. Observe with an x40 objective and estimate the percentage of spermatozoa moving at the following speeds:
Grade 0: No movement at all
Grade 1: Moving with no forward progression
Grade 2: Moving with slow and wandering movement
Grade 3: Moving rapidly in an almost straight line
Grade 4: Moving with high speed in a straight line
Calculate a motility score by adding up the product motility grade and percentage of spermatozoa in that grade. Motility depends upon temperature. At 37°C, only 50% of sperm are motile after 3 hours. At 21°C, 50% are still motile after 7 hours. However, the temperature below 20°C decreases the motility. Artefactual asthenozoospermia can be produced by contamination of the container with water, soap, detergents, or after contact with rubber. Asthenozoospermia caused by cold exposure (<20°C) of the semen sample, infection or fructose deficiency can be easily diagnosed by performing the following simple test:
Exposure to cold: Return of sperm motility after placing the semen sample for 30 min in the incubator is diagnostic of reduced motility due to cold.
Infection: Manifested by the presence of excess white cells or bacteria. Bacterial culture will help. Fructose deficiency: Addition of an equal volume of warm Bakers buffer (3.0g glucose, 0.46g Na2HPO4 7H2O, 0.2g NaCl, 0.01g KH2PO4 and distilled water up to 100 ml) to an aliquot of Human semen on a glass slide will produce a resumption of sperm motility if due to fructose deficiency.
ASSESSMENT OF SPERM MORPHOLOGY
A normal sperm consists of a head and a tail joined together by a short neck. The head is oval in shape and measures 4.5x3x1.5 ȝm while the tail is about 50 ȝm long (10 times the head and neck length). The tail comprises a mid-piece, the principal piece, and a terminal segment. Most of the tail length (90%) is composed of the principal piece. Assessment of morphology can be made in a wet preparation or in a stained smear of semen. As it is difficult to define morphology in a motile sperm, it is better to use a stained smear. For staining, smear is made in the same way as blood smear is made. It can then, be stained by hematoxylin and eosin or Papanicolaou or May-Grunwald-Giemsa stains. The slides are then examined under oil immersion objective of the microscope.
Abnormalities of the head including small, large, tapering, pyriform, amorphous and double heads; of the tail including double, coiled or short tails and of the mid-pieces should be noted. At least 100 spermatozoa must be examined, the percentage of abnormal sperms should be stated and morphological abnormalities described. Also make a note of the presence of white blood cells, epithelial cells, red blood cells, germinal cells, lymphocytes, extraneous particles, protozoa, and bacteria.
REPORTING
Some of the special terms used for reporting the results of Human semen analysis are:
Aspermia: No ejaculate. Oligospermia/Hypospermia: Reduction in volume of ejaculate.
Hyperthermia: Increase in volume of ejaculate.
Oligozoospermia: Low sperm count (<30 million/ml).
Polyzoospermia: High sperm count (>300 million/ml)
Asthenozoospermia: Absence or marked reduction in sperm motility (Motility score <150)
Oligoasthenozoospermia: Low count with low motility.
Necrospermia: Dead sperm
REFERENCE RANGE
Volume 2-6 ml (1-10 ml) Colour Grey-yellow Appearance Opalescent Viscosity Viscous pH 7.2-8.9 Sperm count 60-150 million/ml (Extreme range 30-300 million/ml) Motility >70% at 1 hour and 50% at 3 hours after ejaculation score • Motility Morphology >70% should be morphologically normal.
TESTING FOR ANTI-SPERM ANTIBODIES
Testing for anti-sperm antibodies is as important in the evaluation of infertile males as the semen analysis and individual laboratories can without much difficulty incorporate these tests in their routine work. Agglutination tests, sperm immobilizing antibody tests, testing for cytotoxic antibodies are the various methods for demonstrating sperm antibodies. Procedure
Separation and preparation of donor sperms
1. Layer 2 ml RPMI1 1640 with 5% FCS2 over semen.
2. Incubate at 37°C for 30 minutes.
3. Take off the uppermost 2 ml.
4. Examine under the microscope for motility.
5. Wash once with RPMI 1640 with 5% FCS.
6. Adjust count to 2000/µl.
Testing antisperm agglutinating and immobilising antibodies
1. Inactivate complement in test and normal serum by incubating at 56°C for 30 minutes. Proceed according to Table.
2. Dispense 1 µl normal serum in column A of rows 1 and 2.
3. Dispense 1 µl of each test serum in column A of rows 3 and 4 so two rows are used for each test serum.
4. Prepare doubling dilutions of the test and normal serum in each row with 5% FCS made in RPMI 1640 i.e., 1:2, 1:4, 1:8, 1:16, 1:32.
5. Mix well on a shaker for 2 minutes after adding 1 µl donor sperm in each well.
6. Incubate at 37°C for 30 minutes.
7. Add 2 µl Rabbit complement in rows 2,4,6,8,10 (the crossed rows) in all wells, leaving well µA to see the antisperm immobilizing antibodies.
8. Incubate 37°C for 1 hour.
9. Observe under the microscope for agglutination.
