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Urinalysis & Body Fluids

Microscopic Analysis

We have completed the physical examination of the semen sample. We know if the accessory glands are functioning correctly, but we have yet to address the most important question: What is the status of the spermatozoa themselves?

We now move to the microscopic analysis. This is the main event. This is where we directly assess the cells that are responsible for fertilization. For the patient, this is the part of the report that holds the answers to their most profound questions about fertility

The microscopic examination is a meticulously standardized process, governed by the World Health Organization (WHO) manual. It is a multi-part investigation that evaluates the quantity, the quality of movement, and the structural integrity of the sperm. Each parameter is a critical piece of the puzzle

Part 1: Sperm Motility - The Race to the Egg

This is the first microscopic assessment we perform, and it is the most time-sensitive. We must evaluate motility within 60 minutes of ejaculation, while the sample is at 37°C, because motility is the first parameter to decline. This test assesses the functional capability of the sperm: Can they swim effectively enough to reach the ovum?

  • Methodology
    1. After liquefaction, the sample is gently but thoroughly mixed
    2. A small, standardized volume (e.g., 10 µL) is placed on a clean microscope slide and covered with a coverslip. The depth of the fluid should be standardized (e.g., using a 20 µm deep counting chamber)
    3. The slide is placed on a microscope stage heated to 37°C
    4. Using phase-contrast microscopy at 200x or 400x magnification, at least 200 spermatozoa across several different fields are evaluated and classified
  • WHO Classification of Motility
    • This is the required, standardized terminology
      • Progressive Motility (PR): These are the “effective” sperm. They are moving actively, either in a straight line or in a large circle. They are making forward progress
      • Non-Progressive Motility (NP): These sperm are moving, but they are not going anywhere. This includes sperm swimming in tight small circles, or sperm where only the tail is twitching but the head is stationary
      • Immotile (IM): No movement at all
  • Calculation & Reference Ranges (WHO 5th Edition)
    • The percentage of each category is calculated
    • Total Motility (PR + NP): The lower reference limit is ≥ 40%. This is the total percentage of sperm that are showing any kind of movement
    • Progressive Motility (PR): The lower reference limit is ≥ 32%. This is the more clinically significant number, representing the sperm that can actually travel through the female reproductive tract
  • Clinical Terminology & Significance
    • Asthenozoospermia: This is the term for reduced sperm motility (when the percentage of progressively motile sperm is below the reference range). It is a very common cause of male infertility. It can be caused by problems with sperm maturation in the epididymis, structural defects in the tail, or prolonged abstinence

Part 2: Sperm Concentration & Total Sperm Count - The Quantitative Assessment

Next, we answer the question: “How many sperm are there?” This is a precise quantitative measurement

  • Methodology (Hemacytometer)
    1. The well-mixed semen sample must be diluted. A specialized sperm-diluting fluid (e.g., a solution of sodium bicarbonate and formalin) is used to immobilize the sperm for easy counting. A common dilution is 1:20
    2. The diluted sample is loaded into a hemacytometer (e.g., a Neubauer chamber)
    3. Under the microscope, sperm are counted within a defined area. For sperm, we typically count the 5 “RBC” squares in the central grid
    4. The calculation accounts for the dilution factor and the volume of the area counted to determine the concentration
    • Calculation Example (1:20 dilution, counting 5 RBC squares): (Number of sperm counted / 5) * 20 (dilution factor) * 50 = Sperm/µL. Then convert to millions/mL
  • Parameters & Reference Ranges (WHO)
    • Sperm Concentration: This is the number of sperm per unit volume
      • The lower reference limit is ≥ 15 million spermatozoa per mL.
    • Total Sperm Count: This is the total number of sperm in the entire ejaculate (Concentration x Volume)
      • The lower reference limit is ≥ 39 million spermatozoa per ejaculate.
  • Clinical Terminology & Significance
    • Oligozoospermia: Low sperm concentration (< 15 million/mL)
    • Azoospermia: The complete absence of sperm in the ejaculate. This is a critical finding that must be confirmed by centrifuging the entire sample and examining the pellet
    • Cryptozoospermia: Very few sperm are found, but only after centrifugation of the pellet
    • Post-Vasectomy Analysis: This is a crucial application. The goal here is to confirm azoospermia, indicating a successful procedure. The presence of any sperm, especially motile sperm, indicates the vasectomy was not successful

Part 3: Sperm Morphology - The Structural Integrity Test

This is often the most technically demanding and subjective part of the analysis. It answers the question: “Are the sperm structurally normal?” A normal shape is essential for motility and for penetrating the ovum

  • Methodology
    1. A thin smear of the semen is made on a microscope slide, similar to a blood smear
    2. The slide is air-dried and stained. The gold standard stain is the Papanicolaou (Pap) stain, but other modified stains (e.g., a rapid Wright-Giemsa based stain) are common
    3. Under oil immersion (1000x magnification), at least 200 spermatozoa are evaluated and classified as either “normal” or “abnormal.”
  • The Gold Standard: Kruger’s “Strict” Criteria
    • The WHO has adopted this very stringent set of criteria for what constitutes a “normal” sperm. Even minor deviations result in the sperm being classified as abnormal
    • A “Normal” Spermatozoon has
      • Head: Smooth, oval shape. Length: 4.0-5.0 µm; Width: 2.5-3.5 µm. A well-defined acrosomal region (the cap containing enzymes for fertilization) should comprise 40-70% of the head area
      • Midpiece: Slender, regular, and attached axially to the head. It should be about the same length as the head
      • Tail: The principal piece should be uniform, uncoiled, and approximately 45 µm long (about 10x the head length)
  • Classification of Defects: Abnormal sperm are categorized by the location of their defect(s):
    • Head Defects: Tapered, pyriform (pear-shaped), amorphous (no defined shape), vacuolated (>20% of head area), small or large head, double heads
    • Midpiece Defects: Bent neck (>90 degrees), asymmetrical insertion, thick or irregular midpiece, abnormally thin midpiece
    • Tail Defects: Short, multiple, hairpin, broken, coiled tails. Cytoplasmic droplets larger than 1/3 the head size are also considered abnormal
  • Reference Range (WHO Strict Criteria)
    • The lower reference limit for normal forms is ≥ 4%.
    • Yes, this means that in a “normal” fertile sample, up to 96% of the sperm can be abnormally shaped! This highlights how stringent the criteria are
  • Clinical Terminology & Significance
    • Teratozoospermia: An increased percentage of abnormally shaped sperm (< 4% normal forms). Poor morphology is strongly associated with failure of fertilization

Part 4: “Round Cells” & WBCs - Searching for Inflammation

The semen sample contains cells other than sperm. We must identify and quantify them

  • The “Round Cell” Dilemma: These are non-spermatozoal, round cells. They are either leukocytes (WBCs) or immature germ cells (spermatids, spermatocytes).
  • Differentiating WBCs from Germ Cells
    • This can be difficult with a standard Wright’s stain
    • The definitive method is the peroxidase stain.: Neutrophils are rich in the enzyme peroxidase and will stain dark brown. Immature germ cells are peroxidase-negative
  • White Blood Cells (Leukocytes)
    • Reference Range (WHO): < 1.0 million WBCs per mL.
    • Clinical Terminology & Significance
      • Leukocytospermia: An elevated concentration of WBCs (> 1.0 million/mL). This is a significant finding that indicates infection or inflammation somewhere in the male reproductive tract (e.g., prostatitis, epididymitis)

Part 5: Sperm Vitality - Dead or Alive?

This test is performed only in specific circumstances to answer the question: “Are the immotile sperm dead, or are they alive but just not moving?”

  • When to Perform: This test is only indicated when progressive motility (PR) is very low (e.g., < 30-40%) or when more than 50-60% of the sperm are immotile
  • Methodology (Eosin-Nigrosin Stain)
    • This is a membrane integrity test. A smear is made with a mixture of eosin (a red dye) and nigrosin (a black background stain)
    • Principle: A live sperm has an intact cell membrane and will exclude the eosin dye, remaining unstained (white) against the dark background. A dead sperm has a damaged membrane that allows the eosin to enter, so it stains red or pink.
  • Interpretation: The percentage of live (unstained) sperm is calculated. In a normal sample, the percentage of live sperm should be slightly higher than the percentage of motile sperm
  • Clinical Terminology & Significance
    • Necrozoospermia: A high percentage of immotile sperm are also found to be non-viable (dead)
    • If a sample has a high percentage of immotile sperm but most are alive: (they exclude the dye), this points to a structural problem with the sperm tail (flagellum), rather than a problem with sperm death

Conclusion

The microscopic analysis is a comprehensive evaluation that gives us a complete picture of the sperm’s potential. We know the quantity (concentration), the functional ability (motility), the structural integrity (morphology), and the viability. By combining these findings, we can accurately characterize the nature of a patient’s male factor infertility, providing the clinician with the precise information needed to counsel the patient and plan the appropriate course of treatment