Semen Analysis: Microscopic Examination for Infertility Test

Microscopic evaluation of semen stands out as the paramount test for assessing infertility in men. This meticulous analysis, focusing on sperm morphology, motility, and concentration, provides crucial insights into reproductive health. By scrutinizing these parameters, clinicians can identify potential impediments to successful conception. The microscopic assessment not only gauges sperm quality but also reveals any abnormalities that may hinder fertilization. This method is instrumental in acquiring precise data for developing targeted interventions and personalized treatment strategies to address male infertility.

Sperm Motility

In the initial laboratory evaluation of sperm function within a wet preparation, the primary focus is on sperm motility, which denotes the sperms' ability to move. This motility is crucial for tasks such as penetrating cervical mucus, navigating the fallopian tube, and ultimately fertilizing the ovum. It is noteworthy that only sperms exhibiting rapid progressive motility possess the capability to successfully penetrate the ovum and facilitate fertilization.

Principle

In the assessment of sperm within a wet preparation, both motile and non-motile spermatozoa are quantified across randomly selected fields using a 40× objective. The outcome is then articulated as the percentage of observed motile spermatozoa.

Method

On a glass slide, a drop of semen is carefully deposited and overlaid with a coverslip, which is then encircled with petroleum jelly to avert dehydration. Subsequently, the specimen is examined under a 40× objective lens. A minimum of 200 spermatozoa is meticulously enumerated across multiple microscopic fields. The findings are expressed as a percentage, delineating (a) rapidly progressive spermatozoa, characterized by swift linear forward movement; (b) slowly progressive spermatozoa, displaying gradual linear or non-linear motion, such as crooked or curved trajectories; (c) non-progressive spermatozoa, manifesting tail movement without concomitant forward progress; and (d) immotile spermatozoa, exhibiting a complete lack of movement (in accordance with WHO criteria). Spermatozoa falling within grades (c) and (d) are classified as poorly motile (asthenospermia). Typically, a minimum of ≥25% of sperm demonstrates rapid progressive motility, or alternatively, ≥50% collectively showcase rapid progressive and slow progressive motility.

If the percentage of motile spermatozoa is less than 50%, it is essential to assess the proportion of viable sperm by examining an eosin preparation.

Sperm Viability or Vitality

Principle

A cell with an intact cell membrane, considered vital or viable, will remain unstained as it does not take up eosin Y. Conversely, a non-viable or dead cell, with a compromised cell membrane, will absorb the dye, resulting in a pink-red stain (refer to Figure 1). An additional stain, such as nigrosin, may be applied to color the background material. This test is conducted when motility is found to be abnormal.

Method

1. Mix one drop of semen with one drop of eosin-nigrosin solution and allow it to incubate for 30 seconds.
2. Make a smear from the drop deposited on a glass slide.
3. Air-dry the smear and inspect it under an oil-immersion objective. Sperms appearing white are categorized as live or viable, whereas red sperms are classified as dead or non-viable. A minimum of 200 spermatozoa are scrutinized.
4. Express the findings as a ratio of viable sperms to non-viable sperms, presented as an integer percentage.

Typically, seventy-five percent or more of spermatozoa are considered to be alive or viable under normal circumstances.

Sperm Count

Principle

The sperm count is conducted post-liquefaction using a counting chamber following appropriate dilution, and the total number of spermatozoa is documented in millions per milliliter (10^6/mL).

Method

1. Dilute semen at a ratio of 1:20 with sodium bicarbonate-formalin diluting fluid. To achieve this, take 1 ml of liquefied semen in a graduated tube and fill it with diluting fluid up to the 20 ml mark. Ensure thorough mixing.
2. Place a coverslip over the improved Neubauer counting chamber, filling the chamber with the well-mixed diluted semen sample using a Pasteur pipette. Subsequently, position the chamber in a humid box for 10-15 minutes to allow spermatozoa to settle.
3. Position the chamber on the microscope stage. Utilizing the 20× or 40× objective and lowering the iris diaphragm adequately for optimal contrast, count the number of spermatozoa in four large corner squares. Consider spermatozoa whose heads touch the left and upper lines of the square as 'belonging' to that square.
4. Calculate sperm count per milliliter using the formula:

   

   • Sperm count = Sperms counted × correction factor × 1000 ÷ Number of squares counted × Volume of 1 square
     Sperm count = Sperms counted × 20 × 1000 ÷ 4 × 0.1
     Sperm count = Sperms counted × 50, 000
5. A normal sperm count is equal to or greater than 20 million per milliliter (i.e., ≥ 20 × 10^6/ml). Sperm counts below 20 million per milliliter may be indicative of male infertility.

Sperm Morphology

To create a smear, a glass slide is used to evenly spread a droplet of seminal fluid, followed by staining. The subsequent step involves counting the percentages of normal and abnormal forms of spermatozoa. Staining techniques employed include Papanicolaou, eosin-nigrosin, hematoxylin-eosin, and Rose Bengal-toluidine blue stain. It is essential to count a minimum of 200 spermatozoa under oil immersion, with the recorded results encompassing the respective percentages of normal and abnormal spermatozoa.

Normal Morphology

A spermatozoon comprises three primary components: the head, neck, and tail. The tail is intricately subdivided into the midpiece, main (principle) piece, and end piece (refer to Figure 2 and Box 1 for visual representation).

The head of the spermatozoon exhibits a pear-shaped structure. Predominantly, the nucleus occupies most of the head, characterized by condensed chromatin with scattered areas known as nuclear vacuoles. The anterior two-thirds of the nucleus is enveloped by the acrosomal cap, a flattened, membrane-bound vesicle containing glycoproteins and enzymes. These enzymes play a crucial role in the separation of corona radiata cells and the dissolution of the ovum's zona pellucida during the process of fertilization.

The neck serves as a brief segment connecting the head and the tail of the spermatozoon. Within the neck, the centriole gives rise to the axoneme of the flagellum. The axoneme is comprised of 20 microtubules arranged in a specific pattern, with a central pair surrounded by 9 peripheral doublets, and it is enclosed by condensed fibrous rings.

The middle piece, which constitutes the initial portion of the tail, features a central axoneme surrounded by robust longitudinal fibers. These fibers, in turn, are enveloped by elongated mitochondria that play a vital role in supplying energy for the movement of the tail.

The primary or main piece forms the majority of the tail and is comprised of an axoneme surrounded by nine robust fibers. This central core is further encased by numerous circularly arranged fibrous ribs.

The endpiece, a short and tapering section, is exclusively composed of the axoneme.

Typically, more than 30% of spermatozoa should exhibit normal morphology (according to WHO, 1999). Morphological abnormalities associated with male infertility encompass defects such as a faulty mid-piece leading to reduced motility, an incomplete or absent acrosome resulting in an inability to penetrate the ovum, and a giant head indicating defective DNA condensation.

Abnormal Morphology

The World Health Organization's morphological classification of human spermatozoa, as of 1999, is outlined 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

Round Cells

Upon microscopic examination, round cells may be identified as either white blood cells or immature sperm cells. To distinguish between the two, a special stain, such as peroxidase or Papanicolaou, is necessary. An elevated count of white blood cells exceeding 1 million/ml suggests the presence of an infection. Similarly, the detection of a substantial number of immature sperm cells indicates dysfunction in spermatogenesis at the testicular level.