SEMEN ANALYSIS

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SEMEN ANALYSIS

Semen analysis, also known as a sperm analysis or sperm count, is a laboratory test that assesses various characteristics of a man’s semen, with a primary focus on the quantity and quality of sperm. It is a crucial diagnostic tool used to evaluate male fertility and reproductive health.

Semen is generated during ejaculation and constitutes spermatozoa suspended within a plasma-like fluid. Sperm production primarily occurs in the testis and the epididymis, which contribute only 5% of the total volume. The bulk of semen consists of secretions from the seminal vesicles (46-80%) and the prostate (13-33%). In addition, the bulbourethral and urethral glands contribute around 2-5% of the total volume. These secretions not only impact semen concentration but also influence its role in supporting spermatozoa. During sexual intercourse, each component is released into the posterior urethra through the processes of emission and subsequent ejaculation. The mixing of these components occurs after ejaculation. This complex process is regulated by androgens, androgen binding protein, transferrin, and inhibin. Initially, sperm are non-motile, but they gain motility while traversing the epididymis due to the addition of carnitine, inositol, lipids, and phospholipids. Furthermore, the ability to penetrate eggs is acquired within the epididymis.

The seminal vesicles play a crucial role by providing an energy source in the form of fructose, which enhances spermatozoa motility. They also contribute prostaglandins and fibrinogen-like substances to the seminal fluid. Prostatic fluid, on the other hand, contains a variety of enzymes, spermine (a bacteriostatic substance), citrates, calcium, and zinc. The bulbourethral and urethral glands add mucoproteins and IgA to semen. Therefore, any ailment affecting these elements of the male genital tract can have a profound impact on both semen’s quality and quantity, potentially leading to infertility.

In summary, semen is a multifaceted fluid comprised of diverse secretions from different male reproductive organs. These components are crucial for sperm motility, energy, and fertilization abilities. Any dysfunction or disease within these components can significantly affect semen quality and quantity, potentially leading to infertility.

INDICATIONS FOR SEMEN ANALYSIS

  1. Couples Experiencing Infertility: Semen analysis is often requested when a couple is unable to conceive after a year of regular, unprotected sexual intercourse. It helps determine if male factor infertility is a contributing issue.

  2. Erectile Dysfunction or Ejaculation Problems: Men with conditions such as erectile dysfunction, premature ejaculation, or anejaculation (inability to ejaculate) may undergo semen analysis to assess their ability to produce viable sperm.

  3. History of Reproductive Disorders: If a man has a history of reproductive disorders, such as undescended testicles, testicular surgery, or a family history of fertility issues, semen analysis may be recommended.

  4. Prior Vasectomy or Vasectomy Reversal: Men who have undergone a vasectomy and are considering vasectomy reversal to restore fertility may have semen analysis done to assess sperm presence and quality following the reversal procedure.

  5. Medical Conditions or Treatments: Some medical conditions and treatments can affect sperm production and quality. Semen analysis is conducted for men with conditions like diabetes, thyroid disorders, cancer, or those undergoing chemotherapy, radiation therapy, or certain medications.

  6. Occupational and Environmental Exposures: Individuals who have been exposed to toxins, chemicals, or radiation in their workplace or environment may undergo semen analysis to evaluate the potential impact on their fertility.

  7. Recurrent Pregnancy Loss: In cases of recurrent pregnancy loss, semen analysis may be part of the fertility assessment to rule out male factor infertility as a contributing factor.

  8. Evaluation of Sterility: Semen analysis can be used to confirm sterility in men who have chosen sterilization procedures like vasectomy or as part of the process for certification of sterility in assisted reproductive technology (ART) settings.

  9. Research and Clinical Studies: In research and clinical studies related to male reproductive health, semen analysis is conducted to gather data and assess the effects of specific factors on sperm quality and count.

SAMPLE COLLECTION

The timing of sample collection is crucial as it can significantly impact the quantity and motility of spermatozoa. Typically, a period of abstinence is recommended, with 3-5 days of abstinence considered adequate. To facilitate the process, collecting the specimen in a laboratory setting is often more practical. However, some patients may experience discomfort and stress in a clinical environment, which can affect the quality and quantity of semen. In such cases, it may be more productive for the patient to produce the specimen at home and promptly transport it to the laboratory. Morning collection is preferred to allow sufficient time for analysis.

Masturbation is the preferred method for semen specimen production, but due to psychological or religious reasons, this may not be feasible for some patients. In such instances, coitus interruptus can be an alternative, although it may result in the loss of a portion of the ejaculate. It’s essential for both the patient and the healthcare provider to be aware of this limitation. Condoms should never be used for semen collection during intercourse. For collecting semen, a clean, dry, wide-mouth glass or plastic container should be used, with a lid that is not rubber-lined, as detergents, water, and rubber can be harmful to sperm. The collected specimen should be transported to the laboratory as close to a temperature of 37°C as possible and delivered within two hours.

This information provides guidance on the appropriate indications and methods for semen analysis, ensuring accurate and reliable results.

PHYSICAL EXAMINATION AND SPERM COUNTING

In the process of semen analysis, attention to physical examination and sperm counting is crucial for accurate assessment. Here’s a detailed breakdown of the steps involved:

Physical Examination:

  • Volume:

The first step in semen analysis is to transfer the collected semen into a meticulously clean graduated small cylinder. In this container, you should carefully measure and record the volume of the ejaculate. This provides an initial assessment of the amount of semen produced by the individual.

  • Color:

After measuring the volume, it’s important to observe and record the color of the semen. The color of normal human semen can vary, but typically falls within a range of white to grayish-white. Any unusual coloration should be noted, as it could be indicative of potential health issues or other factors that require further investigation.

  • Appearance:

The next aspect to assess is the appearance of the semen. Upon ejaculation, human semen often forms a gel-like clot, which is a normal reaction. However, this clot should typically liquefy within a period of 5-20 minutes. It is crucial to record whether the semen has already liquefied by the time it’s brought to the workbench or if it remains in a gel-like state. Any absence of liquefaction should be noted.

  • pH:

Another critical parameter in semen analysis is the pH level. The pH of semen should be assessed and recorded. Typically, the pH of human semen falls within a range of about 7.2 to 7.8. Deviations from this range could be indicative of underlying health issues or other factors that require consideration.

Sperm Counting:

  1. Visual Assessment: Place a drop of semen on a clean glass slide and gently cover it with a cover slip. Examine the slide under the high-power objective of a microscope to visually assess the sperm count and determine if any dilution is necessary.
  2. Dilution: Prepare a diluent by dissolving 5 g of sodium bicarbonate, 1 ml of 35% formalin, and distilled water to a total volume of 100 ml. To stain the sperm, you can add 5 ml of a saturated aqueous solution of gentian violet. This fluid immobilizes spermatozoa and facilitates counting. Normally, a 1 in 20 dilution is made by adding 50 μl of well-mixed and liquefied semen to 950 μl of diluent using a Sahli pipette. However, a 1 in 10 dilution is recommended, and a 1 in 50 dilution may be necessary if the sperm count appears to be high.
  3. Counting Procedure: An Improved Neubauer chamber (Haemocytometer) is employed for counting. Ensure that both the chamber and the cover slip are cleaned with distilled water and dried before use. The cover slip is then pressed onto the central area until air is expelled, and birefringent rings appear on the sides. Carefully mix the diluted semen and charge the chamber using a Pasteur pipette. Examine the chamber using a x10 objective of a microscope. Count sperm in the four large corners and one large central square (WBC counting area). It is essential not to count loose tails and germinal cells. A minimum of 200 spermatozoa must be counted, and if this count is not met in the initial five squares, additional squares should be counted. These steps ensure a comprehensive assessment of semen, providing vital information for fertility evaluation and related procedures.
  4. Calculation
      • Sperm count (million/ml) = CxDx1000/V
      • C = Count in 5 large squares
      • D = Dilution factor
      • V = Vvolule of 5 large s1ures
      • 1000 = To convert mm3 into ml

ASSESSMENT OF SPERM MOTILITY

Assessing sperm motility is a critical step in semen analysis, and it should be conducted at various time intervals after sample production. It’s essential to note that sperm require at least 10 μm of depth for free movement. Here’s how to perform the assessment:

Method 1:

  1. Preparation of Slide: Take a drop of well-mixed undiluted semen and place it on a warm, clean slide. Gently cover it with a cover slip. Allow the slide to rest on the microscope stage or bench until the “streaming” of the semen stops.
  2. Microscopic Examination: Examine the slide under the microscope. In at least five different fields, count both motile and immotile sperm, with a minimum count of 200 spermatozoa. This counting process should be performed in duplicate, and the average should be recorded. Only forward movement of the sperm is considered positive.
  3. Percentage Motility Calculation: Calculate the percentage motility. The sperm count can be determined using the following formula:

Sperm Count =  Sperm count/ml X % motility semen volume/100

Method 2 (Alternative Procedure for Motility Grading):

  1. Post-Collection Handling: Approximately 30 minutes after collection, transfer the semen into a capped tube. Thoroughly mix by gently inverting the tube several times.
  2. Slide Preparation: Pipette one drop of semen onto a clean glass slide and place a clean cover slip over it.
  3. Motility Grading: Observe the slide using a x40 objective on a microscope. Estimate the percentage of spermatozoa moving at different speeds, assigning them grades as follows:
      • Grade 0: No movement at all
      • Grade 1: Moving but 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
  4. Motility Score Calculation: Calculate a motility score by adding up the product of the motility grade and the percentage of spermatozoa in that grade.

For example, if 20% of sperm are Grade 3 (rapidly moving in an almost straight line) and 40% are Grade 2 (moving with slow and wandering movement), the motility score would be (20% x 3) + (40% x 2) = 1.2 + 0.8 = 2.0.

Sperm Motility
Sperm Motility

These methods ensure an accurate assessment of sperm motility, providing valuable information for fertility evaluation and related procedures.

SPERM MOTILITY AND RELATED FACTORS

Assessing sperm motility is a crucial aspect of semen analysis, and a normal motility score for spermatozoa is typically considered to be ≥150. However, it’s important to note that sperm motility can be influenced by various factors, and the temperature plays a significant role in this regard.

At 37°C, approximately 50% of sperm are still motile after 7 hours. Lower temperatures, particularly below 20°C, can significantly decrease sperm motility. Certain conditions can lead to what is referred to as artefactual asthenozoospermia, which can result from contamination of the semen container with water, soap, detergents, or contact with rubber.

Diagnosing asthenozoospermia, whether due to cold exposure, infection, or fructose deficiency, can be achieved through simple tests:

  1. Exposure to Cold: If reduced motility is suspected due to cold exposure (temperatures below 20°C), placing the semen sample in an incubator for 30 minutes and observing the return of sperm motility can be diagnostic.
  2. Infection: The presence of excessive white blood cells or bacteria in the sample may indicate infection. To confirm, a bacterial culture can be conducted.
  3. Fructose Deficiency: If fructose deficiency is suspected, adding an equal volume of warm Baker’s buffer (containing 3.0g glucose, 0.46g Na2HPO4·7H2O, 0.2g NaCl, 0.01g KH2PO4, and distilled water to make 100 ml) to an aliquot of semen on a glass slide can lead to a resumption of sperm motility.

Understanding and addressing these factors are essential in the evaluation of sperm motility and fertility.

ASSESSMENT OF SPERM MORPHOLOGY AND REPORTING

Evaluating sperm morphology is a critical component of semen analysis, as it provides insights into the structure and shape of sperm. A typical sperm consists of a head, a neck, and a tail. The head is oval-shaped, measuring approximately 4.5x3x1.5 μm, while the tail is about 50 μm in length, which is roughly ten times the length of the head and neck combined. The tail is further divided into a mid-piece, the principal piece, and a terminal segment, with the principal piece comprising the majority of the tail length (approximately 90%).

Sperm morphology assessment can be conducted using a wet preparation or a stained smear of semen. To better define morphology, a stained smear is often preferred. Smears can be made in a manner similar to creating blood smears and can then be stained with substances like haematoxylin and eosin, Papanicolaou, or May-Grunwald-Giemsa stains. Slides are subsequently examined under an oil immersion objective of the microscope. Abnormalities in the head (including small, large, tapering, pyriform, amorphous, and double heads), the tail (including double, coiled, or short tails), and the mid-pieces should be noted. A minimum of 100 spermatozoa must be examined, and the percentage of abnormal sperms should be reported, along with a description of the observed morphological abnormalities. Additionally, any presence of white blood cells, epithelial cells, red blood cells, germinal cells, lymphocytes, extraneous particles, protozoa, and bacteria should be noted.

Sperm Morphology
Sperm Morphology

In reporting the results of semen analysis, specific terms are used to describe the findings, including:

  • Aspermia: No ejaculate is present.
  • Oligospermia/Hypospermia: There is a reduction in the volume of ejaculate.
  • Hyperspermia: There is an increase in the volume of ejaculate.
  • Oligozoospermia: The sperm count is low (<30 million/ml).
  • Polyzoospermia: The sperm count is high (>300 million/ml).
  • Asthenozoospermia: There is an absence or marked reduction in sperm motility (Motility score <150).
  • Oligoasthenozoospermia: Both the sperm count and motility are low.
  • Necrospermia: The sperm are dead.

This comprehensive assessment of sperm morphology and reporting is essential in fertility evaluation and diagnosis.

REFERENCE RANGE FOR SEMEN ANALYSIS:

  • 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 (with an extreme range of 30-300 million/ml)
  • Motility:
      • 70% at 1 hour after ejaculation
      • 50% at 3 hours after ejaculation
      • Motility score should be ≥150
  • Morphology:
      • 70% should be morphologically normal

Important Notes:

    • Retrograde ejaculation, where semen is directed backward into the urinary bladder, can be a significant cause of aspermia (lack of ejaculation). In such cases, it is essential to immediately collect and examine a urine specimen for the presence of spermatozoa.
    • Not all immotile sperm are necessarily dead. It is important to differentiate between asthenozoospermia (reduced sperm motility) and necrospermia (dead sperm). To distinguish between the two, mix a drop of semen with a drop of 0.5% yellow eosin in distilled water on a glass slide, place a cover slip, and examine it under a microscope. Dead spermatozoa will take on a pink-yellow color, while immotile but living sperm remain unstained.
    • In the evaluation of infertility, it is recommended to perform semen analysis on three separate occasions, with a gap of 2-3 weeks between any two analyses. This helps provide a more accurate assessment of the patient’s fertility status.

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