MICROSCOPIC EXAMINATION OF SEMEN FOR INVESTIGATION OF INFERTILITY

Published in Clinical Pathology
Monday, 14 August 2017 22:02
The most important test in semen analysis for infertility is microscopic examination of the semen.
 
SPERM MOTILITY
 
The first laboratory assessment of sperm function in a wet preparation is sperm motility (ability of the sperms to move). Sperm motility is essential for penetration of cervical mucus, traveling through the fallopian tube, and penetrating the ovum. Only those sperms having rapidly progressive motility are capable of penetrating ovum and fertilizing it.
 
Principle: All motile and non-motile sperms are counted in randomly chosen fields in a wet preparation under 40× objective. Result is expressed as a percentage of motile spermatozoa observed.
 
Method: A drop of semen is placed on a glass slide, covered with a coverslip that is then ringed with petroleum jelly to prevent dehydration, and examined under 40× objective. Atleast 200 spermatozoa are counted in several different microscopic fields. Result is expressed as a percentage of (a) rapidly progressive spermatozoa (moving fast forward in a straight line), (b) slowly progressive spermatozoa (slow linear or non-linear, i.e. crooked or curved movement), (c) non-progressive spermatozoa (movement of tails, but with no forward progress), and (d) immotile spermatozoa (no movement at all) (WHO critera). Sperms of grades (c) and (d) are considered to be poorly motile (asthenospermia). Normally, ≥ 25% of sperms show rapid progressive motility, or ≥ 50% of sperms show rapid progressive and slow progressive motility.
 
If the proportion of motile spermatozoa is < 50%, then proportion of viable sperms should be determined by examining an eosin preparation.
 
SPERM VIABILITY OR VITALITY
 
Principle: A cell with intact cell membrane (a vital or viable cell) will not take up the eosin Y and will not be stained, while a non-viable or dead cell will have damaged cell membrane, will take up the dye, and will be stained pink-red (Figure 832.1). Another stain (e.g. nigrosin) may  be used to stain the background material. The test is performed if motility is abnormal.
 
Figure 832.1 Eosin nigrosin stain
Figure 832.1 Eosin-nigrosin stain. Dead sperms are stained pink-red, while live sperms are stained white
 
Method
 
  1. Mix one drop of semen with 1 drop of eosin-nigrosin solution and incubate for 30 seconds.
  2. A smear is made from a drop placed on a glass slide.
  3. The smear is air-dried and examined under oilimmersion objective. White sperms are classified as live or viable, and red sperms are classified as dead or non-viable. At least 200 spermatozoa are examined.
  4. The result is expressed as a proportion of viable sperms against non-viable as an integer percentage.
 
Seventy-five percent or more of sperms are normally live or viable.
 
SPERM COUNT
 
Principle: The sperm count is done after liquefaction in a counting chamber following dilution and the total number of spermatozoa is reported in millions/ml (106/ml).
 
Method
 
  1. Semen is diluted 1:20 with sodium bicarbonateformalin diluting fluid (Take 1 ml liquefied semen in a graduated tube and fill with diluting fluid to 20 ml mark. Mix well).
  2. A coverslip is placed over the improved Neubauer counting chamber and the counting chamber is filled with the well-mixed diluted semen sample using a Pasteur pipette. The chamber is then placed in a humid box for 10-15 minutes for spermatozoa to settle.
  3. The chamber is placed on the microscope stage. Using the 20× or 40× objective and iris diaphragm lowered sufficiently to give sufficient contrast, number of spermatozoa is counted in 4 large corner squares. Spermatozoa whose heads are touching left and upper lines of the square should be considered as ‘belonging’ to that square.
  4. Sperm count per ml is calculated as follows:

    Sperm count =                Sperms counted × correction factor             × 1000
                              Number of squares counted × Volume of 1 square
                           = Sperms counted × 20 1000
                                        4 × 0.1
                           = Sperms counted × 50, 000

  5. Normal sperm count is ≥ 20 million/ml (i.e. ≥ 20 × 106/ml). Sperm count < 20 million/ml may be associated with infertility in males.
 
SPERM MORPHOLOGY
 
A smear is prepared by spreading a drop of seminal fluid on a glass slide, stained, and percentages of normal and abnormal forms of spermatozoa are counted. The staining techniques used are Papanicolaou, eosinnigrosin, hematoxylin-eosin, and Rose Bengal-toluidine blue stain. Atleast 200 spermatozoa should be counted under oil immersion. Percentages of normal and abnormal spermatozoa should be recorded.
 
Normal morphology: A spermatozoon consists of three main components: head, neck, and tail. Tail is further subdivided into midpiece, main (principle) piece, and end piece (Figure 832.2 and Box 832.1).
 
Figure 832.2 Morphology of spermatozoa
Figure 832.2 Morphology of spermatozoa
 
Head is pear-shaped. Most of the head is occupied by the nucleus which has condensed chromatin and few areas of dispersed chromatin (called nuclear vacuoles). The anterior 2/3rds of the nucleus is surrounded by acrosomal cap. Acrosomal cap is a flattened membranebound vesicle containing glycoproteins and enzymes. These enzymes are required for separation of cells of corona radiata and dissolution of zona pellucida of ovum during fertilization.
 
Neck is a very short segment that connects the head and the tail. Centriole in the neck gives rise to axoneme of the flagellum. Axoneme consists of 20 microtubules (arranged as a central pair surrounded by 9 peripheral doublets) and is surrounded by condensed fibrous rings.
 
Middle piece is the first part of the tail and consists of central axoneme surrounded by coarse longitudinal fibers. These are surrounded by elongated mitochondria that provide energy for movement of tail.
 
Principle or main piece constitutes most of the tail and is composed of axoneme that is surrounded by 9 coarse fibers. This central core is surrounded by many circularly arranged fibrous ribs.
 
Endpiece is the short tapering part composed of only axoneme.
 
Normally, > 30% of spermatozoa should show normal morphology (WHO, 1999). The defects in morphology that are associated with infertility in males include defective mid-piece (causes reduced motility), an incomplete or absent acrosome (causes inability to penetrate the ovum), and giant head (defective DNA condensation).
 
Box 832.1 Normal sperm morphology
• Total length of sperm: About 60 μ
• Total length of sperm: About 60 μ
• Head:
   – Length: 3-5 μ
   – Width: 2-3 μ
   – Thickness: 1.5 μ
• Neck: Length: 0.3 μ
• Middle piece:
   – Length: 3-5 μ
   – Width: 1.0 μ
• Principal piece:
   – Length: 40-50 μ
   – Width: 0.5 μ
• End piece: 4-6 μ
 
Abnormal morphology (Figure 832.3): WHO morphological classification of human spermatozoa (1999) is given below:
 
  1. Normal sperm
  2. Defects in head:
    • Large heads
    • Small heads
    • Tapered heads
    • Pyriform heads
    • Round heads
    • Amorphous heads
    • Vacuolated heads (> 20% of the head area occupied by vacuoles)
    • Small acrosomes (occupying < 40% of head area)
    • Double heads
  3. Defects in neck:
    • Bent neck and tail forming an angle >90° to the long axis of head
  4. Defects in middle piece:
    • Asymmetric insertion of midpiece into head
    • Thick or irregular midpiece
    • Abnormally thin midpiece
  5. Defects in tail:
    • Bent tails
    • Short tails
    • Coiled tails
    • Irregular tails
    • Multiple tails
    • Tails with irregular width
  6. Pin heads: Not to be counted
  7. Cytoplasmic droplets
    • > 1/3rd the size of the sperm head
  8. Precursor cells: Considered abnormal
 
Figure 832.3 Abnormal morphological sperm forms
Figure 832.3 Abnormal morphological sperm forms: (1) Normal sperm, (2) Large head, (3) Small head, (4) Tapered head, (5) Pyriform head, (6) Round head, (7) Amorphous head, (8) Vacuoles in head, (9) Round head without acrosome, (10) Double head, (11) Pin head, (12) Round head without acrosome and thick midpiece, (13) Coiled tail, and (14) Double tail

ROUND CELLS
 
Round cells on microscopic examination may be white blood cells or immature sperm cells. Special stain (peroxidase or Papanicolaou) is required to differentiate between them. White blood cells >1 million/ml indicate presence of infection. Presence of large number of immature sperm cells indicates spermatogenesis dysfunction at the testicular level.

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