Semen Analysis Using Sperm Class Analyzer (SCA v5) for Computer Assisted Semen Analysis (CASA)

Abstract

Semen analysis yields useful information about the fertility status of an individual and is one of the first steps in the diagnosis of male factor infertility. This test provides an assessment of fertility potential, sperm production, functioning of accessory genital tract glands, and ejaculatory capability.

Appendices

10 Appendix 1: Center the Phase Contrast Condenser

• 1. Rotate the phase contrast turret until the letter “O” can be seen through the central window, and open the aperture iris diaphragm fully.

• 2. Place a specimen on the stage, bring the 10× objective into the light path, and focus on the specimen (EN slide).

  Note: If an auxiliary lens or high/low magnification selector is provided with the microscope used, set it to the position for the 10× objective.

• 3. Top down (close to right) the field iris diaphragm of the microscope.

• 4. While looking through the eyepiece, move the condenser up and down with the condenser height adjustment to focus on the image of the field iris diaphragm.

• 5. Widening the diameter of the field iris diaphragm progressively, adjust the condenser centering knobs (front knobs) to bring the diaphragm image into the center of the field of view. When the polygonal image of the iris diaphragm becomes inscribed in the field, slightly increase the diameter of the field iris diaphragm until it is just outside the field of view.

11 Align the Phase Annulus and Light Annulus

• 1. Swing the objective of your choice into the light path.

• 2. Rotate the phase contrast turret until the number corresponding to the objective magnification is seen through the central window.

• 3. Focus on the specimen (E/N slide).

  Note: In the case that the microscope is provided with the auxiliary lens system or high/low magnification selector lever, set it to the position for the objective magnification used.

• 4. Remove one of the eyepieces and insert the centering telescope CT-5 into the eyepiece tube.

• 5. Rotate the top lens assembly of the CT-5 to bring (light annulus in the objective) into focus.

• 6. Rotate the two light annulus centering knobs (back knobs) of the condenser until light annulus and phase annulus are concentric and superimposed.

12 Appendix 2: Calibration of Microscope Reticle

• A. The microscope reticle is calibrated using a stage micrometer.

• B. Load the micrometer onto the stage and focus and line up the micrometer with the reticle grid.

• C. Count the micrometer lines that measure across 10 squares of the reticle using the 10× and 20×.

  Use the following formula to calculate the microscope conversion factors:

  20 μ chamber thickness

  (D ²) the number of increments per reticule square times 0.01 mm and squared:

  $$ \frac{1,000,000}{T\times {D}^2}=\mathrm{microscope}\ \mathrm{factor} $$

Example: Using the 20× Lens

• 57 increments/10 squares = 5.7 increments/squares.

• 1 increment equals 1/100 mm.

• 5.7 × 0.01 mm = 0.057 mm.

• 0.057 mm × 1000 = 57 μ (D).

• D = 57 D ² = 57 × 57 = 3249.

• $$ \frac{1,000,000}{(20)\times 3249} = \frac{1,000,000}{64,980}\kern0.5em = 15.39\left(\mathrm{factor}\right) $$

13 Appendix 3: Retrograde Ejaculate Processing for Routine Semen Analysis

• A. The patient should collect the urine sample directly after the semen sample in 9 mL of HTF media.

• B. When urine specimen arrives to the laboratory, measure the total volume of the urine (and HTF) and record on the pink worksheet.

• C. Aliquot the specimen into sterile conical tubes (with lids) and centrifuge at 1600 rpm for 10 min.

• D. Pour off (or aliquot) the urine supernatant into a waste container.

• E. Reconstitute the first tube with 1–2 mL of HTF. Transfer 1–2 mL from the first tube to second tube and reconstitute. Continue this process until all the tubes are reconstituted.

• F. Do the semen analysis on the last reconstituted tube.

• G. When entering the volume of specimen into the CASA (SCA), enter the reconstituted volume of 1–2 mL depending on how much HTF you originally added.

• H. Base your calculations on the reconstituted volumes of 1–2 mL.

• I. Please put the reconstituted volume in the volume field and comment on the original urine volume.

  For example: Semen suspended in 50 mL of urine. Specimen spun and resuspended in HTF.

• J. Process the reconstituted specimen for the routine analysis as per Routine Analysis procedure.

14 Appendix 4: Sperm Morphology Differentials in Severely Oligozoospermic Specimens

• A. In patients with poor sperm counts (<10 × 10⁶/mL), centrifuge the semen sample prior to making two long smear slides. Be sure the rest of the semen analysis is completed prior to centrifugation.

  Note: Make two long slides using the unspun specimen as well.

• B. Centrifuge the specimen at 1600 rpm for 7 min. Remove the supernatant (seminal plasma) and resuspend the sperm pellet in 0.3–0.5 mL of PBS (1×) solution. Mix gently. Make a set of long slides on this centrifuged specimen for morphology differentiation.

• C. When reading the differential, save one spun and unspun slide for the slide archives. Please make an attempt to read the differential. You may score 25 or 50 sperm as a percentage for the very low specimens. If <100 sperm are scored, make a note on the report form indicating how many spermatozoa were counted.

  Note: Do not include the round cells as part of the differential.

15 Appendix 5: Manual Counts and Motilities

6.1 15.1 Manual Counts: Using the Sperm Counting Chamber

Count a minimum of 100 cells this includes motile and nonmotile sperm (this may take more than one field; insure the fields have similar concentration).

• 1. Using the 20× objective:

  $$ \begin{array}{l}\mathrm{Concentration} = \frac{\mathrm{Average}\ \#\ \mathrm{Cells}}{\mathrm{Number}\;\mathrm{Fields}}\times \left(20\times \mathrm{Row}\;\mathrm{factor}\right)\\ {}\kern7.5em =\dots \times {10}^6/\mathrm{mL}\end{array} $$

  Note: See Appendix 2.

6.2 15.2 Manual Motility: Using the Sperm Counting Chamber

When doing a manual count which includes motile and nonmotile sperm, without changing the field, count only the motile sperm and calculate the percent motility:

$$ \frac{\#\ \mathrm{motile}\ \mathrm{cells}\ \mathrm{counted}}{\mathrm{Total}\ \#\ \mathrm{of}\ \mathrm{cells}\ \mathrm{counted}}\times 100 = \%\ \mathrm{motile}\ \mathrm{sperm} $$

6.2.1 15.2.1 Round Cell Estimation (RC)

• 1. Using the sperm counting chamber at 20×: Count the number of round cells in 200 boxes (two fields):

  $$ \#\ \mathrm{R}\mathrm{C}\ \mathrm{C}\mathrm{ounted} = \frac{\mathrm{Average}\ \#\mathrm{R}\mathrm{C}}{\mathrm{Number}\ \mathrm{of}\ \mathrm{Fields}}\times \left(20\times \mathrm{Row}\ \mathrm{Factor}\right)/100\times {10}^6/\mathrm{mL} $$

16 Appendix 6: Viscosity Treatment System

7.1 16.1 VTS Procedure

• 1. Purpose: This proteolytic enzyme treatment system will be used when a freshly collected semen specimen obtained by masturbation fails to undergo liquefaction after 30 min of incubation at 37 °C. Its use is intended to assist in sample preparation prior to analysis.

• 2. Reagents: Viscosity Treatment System

  20 vials (treatments) of proteolytic enzyme contain 5 mg chymotrypsin per vial (treatment).

  The reagent is stored at −20 °C until use. See package for expiration.

• 3. Procedure:

  • A. Remove one vial (proteolytic enzyme) from the −20 °C freezer.

  • B. Tap the vial on the countertop to dislodge any powder that may be adhering to the vial cap.

  • C. Add the contents of the vial (proteolytic enzyme) to the semen specimen and gently mix the specimen.

    Note: VTS should not be used on specimens with a total volume <0.5 mL.

  • D. Incubate the specimen at 37 °C until it liquefies (~10 min).

  • E. Once liquefaction has occurred, perform semen analysis immediately.

    Note: If the analysis is not performed immediately, the enzyme should be neutralized by adding a small amount of sperm washing media (0.5 mL).

  • F. The semen specimen should be processed according to the routine semen analysis procedure.

  • G. Make note on the patient report form that the specimen had high viscosity and was treated with VTS.

17 Appendix 7: Sperm Count Only

• 1. Some physicians order a sperm count only to check if their patient has sperm production.

• 2. When the specimen arrives in the laboratory, measure the total volume and record on the patient report form.

• 3. 5 μL of well-mixed semen loaded onto a sperm counting chamber and at least 200 sperm scored per routine procedure. The result is recorded on the patient report form.

• 4. A manual motility is performed per routine procedure and recorded on the patient report form.

• 5. The sperm concentration (M/mL) and total sperm count (M) is reported under the appropriate field. Sperm motility is reported as a comment.