Semen analysis

• Semen Composition

  • Spermatozoa - 5%
  • Seminal Fluid - 60% to 70%
  • Prostate Fluid - 20% to 30%
  • Bulbourethral Glands - 5%
• Testes


  • Paired glands in the scrotum that contain the seminiferous tubules for sperm secretion
  • Where spermatogenesis occurs
• Epididymis


  • Where the sperm mature and develop flagella
• Seminal vesicles


  • Produce most of the fluid present in semen (60% - 70%)
  • Fluid is the transport medium for the sperm
  • High concentration of fructose (source of sperm cell's energy) and flavin (chemical responsible for grayish color of sperm cell)
• Prostate Gland


  • Aims in propelling the sperm through the urethra by contractions during ejaculation
  • Produces the acidic fluid which comprises 20% to 30% of the semen volume
• Bulbourethral glands


  • Contribute about 5% of the fluid volume in the form of a thick, alkaline mucus
• SPECIMEN COLLECTION

  1. Masturbation

  2. Nonlubricant-containing rubber or polyurethane condoms when masturbation isn't possible

  3. Collected following a period of sexual abstinence of at least 2 days to not more than 7 days

  4. Two or three samples collected not less than 7 days or more than 3 weeks for fertility testing

  5. Warm sterile glass or plastic containers

  6. Kept at room temperature and delivered to the laboratory within one hour of collection

  7. Record patient's name, birthdate, period of sexual abstinence, completeness of the sample, difficulties with collection, times of specimen collection and receipt

  8. Specimen awaiting analysis should be kept at 37°C
• Standard precautions must be observed at all times
• Discarded as biohazardous waste
• Use of sterile materials and techniques
• Normal semen appearance

  Gray to white color, appears translucent, and has a characteristic musty odor
• Almost clear specimen


  Indicates very low sperm concentration
• Increased turbidity


  Indicates (+) for WBCs and infection within the reproductive tract
• Varying amount of red coloration


  Indicates (+) for RBCs
• Yellow coloration


  Indication for urine contamination, specimen collection following prolonged abstinence, and/or medications
• LIQUEFACTION

  1. Fresh semen specimen is clotted and should liquefy within 30-60 minutes after collection

  2. Failure of liquefaction may be caused by a deficiency in prostatic enzymes

  3. Analysis of the specimen cannot begin until liquefaction has occurred

  4. Equal volume of physiologic Dulbecco's phosphate-buffered saline or proteolytic enzymes such as alphachymotrypsin or bromelain may be added to induce liquefaction
• Normal semen volume

  Between 2 and 5 mL
• Increased volume


  Brought by periods of extended abstinence
• Decreased volume


  • Due to (+) infertility, improper functioning of one of the semen-producing organs, primarily the seminal vesicles
  • Incomplete specimen collection must also be considered
• Normal semen viscosity


  Easily drawn into a pipette and form small discrete droplets that doesn't appear clumped or stringy
• Highly viscous, abnormal semen

  Droplets that form threads longer than 2 cm
• Manner of reporting semen viscosity

  0 (watery) to 4 (gel-like), low, normal, or high
• Normal semen pH

  Alkaline (7.2-8.0)
• Increased pH


  Indicates (+) infection within the reproductive tract
• Decreased pH


  Indicates increased prostatic fluid, ejaculatory duct obstruction, or poorly developed seminal vesicles
• Reference values for sperm concentration

  >20 to 250 million sperm/mL
• Borderline sperm concentration

  Between 10 and 20 million/mL
• Normal total sperm count

  Greater than 40 million per ejaculate (20 million per milliliter x 2 mL)
• Sperm concentration measurement

  1. Using the Neubauer counting chamber

  2. 1:20 is the most commonly used dilution

  3. Dilution of semen is essential

  4. Traditional Diluting Fluid is sodium bicarbonate + formalin

  5. Counted in the four corner and center squares of the large center square, similar to a manual RBC count

  6. Both sides of the hemocytometer (counting chamber) are loaded and allowed to settle for 3-5 minutes, then counted, and the counts should agree within 10

  7. Average of the two counts is used in calculation

  8. Only full developed sperm should be counted
• Count greater than 1 million leukocytes per milliliter


  Associated with inflammation or infection of the reproductive organs → infertility
• (+) more than 1 million spermatids per milliliter


  Indicates disruption of spermatogenesis
• Sperm concentration calculation

  When using the 1:20 dilution and counting the five squares (RBCs) in the large center square, the number of sperm can be multiplied by 1,000,000 to equal the sperm concentration per milliliter
• Sperm concentration reporting

  In million per milliliter
• Normal sperm motility

  Minimum motility of 50% with a rating of 2.0 after 1 hour
• Sperm morphology

  • Oval-shaped head approximately 5 µL long and 3 µL wide
  • Long, flagellar tail approximately 45 µL long
  • Acrosomal Cap contains enzyme, critical to ovum penetration and should encompass approximately half of the head and cover approximately two thirds of the sperm nucleus
  • Neckpiece attaches the head to the tail and the midpiece
  • Midpiece is approximately 7 µL long and is the thickest part of the tail because it is surrounded by a mitochondrial sheath that produces the energy required by the tail for motility
• Sperm morphology evaluation

  1. Can be evaluated at room temperature or 37°C; sample should be incubated at this temperature and preparation made with prewarmed slides and cover slips

  2. Evaluated from a thinly smeared, stained slide under oil immersion

  3. At least 200 sperm should be evaluated and the percentage of abnormal sperm

  4. Kruger's Strict Criteria: include measuring head, neck, and tail size, measuring acrosome size, and evaluating for presence of vacuoles
• Normal sperm morphology


  • Routine Criteria: >30% normal forms
  • STRICT Criteria: >14% normal forms
• Calculating Round Cells

  1. Counting the number of spermatids or leukocytes seen in conjunction with 100 mature sperm

  2. N is the number of spermatids/neutrophils counted per 100 mature sperm and S is the sperm concentration in millions per milliliter

  3. Used when counting cannot be performed during hemocytometer count and to verify counts performed by hemocytometer
• Greater than 1 million WBCs per milliliter per ejaculate


  Indicates an inflammatory condition
• Decreased sperm vitality


  (+) normal sperm concentration with markedly decreased motility