Spermac Protocol to Assess Human Sperm Acrosome Status

For the rapid, determination of acrosome-reaction in sperm smears. This web site may be referenced as:

Chan PJ, Corselli J., Patton W., Jacobson J. The Spermac Stain. Loma Linda University, CA, USA, (1996) /lluhc/fertility/

INTRODUCTION

Spermac provides a good definition of the staining for sperm as a clinical assessment of acrosome reacted or intact sperm. The stains are evenly applied and provide constant quality which insures reliability and satisfactory staining for the sperm. Spermac can also be used for sperm morphology assessment. Morphology, acrosome reaction, in conjunction with other parameters of semen analysis provide a clinical marker in predicting fertilization rates. For the human sperm, spermac stains the postacrosomal region and the nuclear portion of the head -RED, the acrosome, midpiece and tail -GREEN. This stain permits effortless determination of acrosome-reacted sperm. A stained slide will be transparent with only a very slight hint of green hue. If the slide is dark green, the slide has been dried too long prior to fixing.

REAGENTS

For in vitro diagnostic use.

1. Fixative (clear liquid) - Formalin based (listed carcinogen)
   
   
2. Stain A (red liquid)
   
   
3. Stain B (yellow liquid)
   
   
4. Stain C (green liquid)

QUALITY CONTROL

1. Store all reagents at room temperature (15-30^oC).
   
   
2. Storage must not exceed expiration date on box label. Reagents are stable for 3 years from date of manufacture.
   
   
3. Indications of deterioration: mold growth; the cells on a slide of a correctly prepared differential smear appear pale when stained.
   
   
4. Before use, check the stains for precipitation or discoloration.

SPECIMEN COLLECTION AND PREPARATION

Human sperm: Use semen collected by one of the methods as outlined in the semen collection procedure. Wear gloves and labcoat when handling semen specimens and treat as if they are highly contagious. Use sterile techniques and sterile disposable items when handling the specimens.

PROCEDURE

1. It is important that the semen is diluted 1:1 with an aqueous solution of sperm washing medium or saline and used immediately to make the smear.
   
   
2. Make a feathered-edge semen smear on a labeled glass slide.
   
   
3. Air dry smears for 10 minutes. DO NOT dry the slides over 30 minutes.
   
   
4. Pipet a few drops of fixative on to the edge of the smear, tilt the glass slide, and allow the fixative to run over the smear.
   
   
5. Place in a horizontal position for 5 mins to 60 mins.
   
   
6. Wash slides by dipping once in tap water. Drain excess.
   
   
7. Pipet a few drops of red-colored Stain A on to the edge of the smear, tilt the slide, and allow the stain to flow down over the smear. Set this aside horizontally for 1 min. We have found that an extra 10 to 20 seconds helps bring out the beautiful red counterstaining.
   
   
8. Dip the slides in a cup of tap water and drain excess.
   
   
9. Pipet almost colorless Stain B on to the edge of the smear, tilt the slide, and let the stain flow down over the smear. Then keep the slides in a horizontal position for 1 min.
   
   
10. Dip the slides in a cup of clean tap water and drain excess.
    
    
11. Pipet dark green Stain C on to the edge of the smear, tilt, and flood the smear for 1 min. Do not stain over 1 minute or you will have very dark slides.
    
    
12. Dip slides in tap water and drain excess.
    
    
13. Air-dry the slides and observe under oil immersion at 400 X objective power.
    
    
14. Score normal (acrosomal intact) sperm as those with green acrosomal and pink postacrosomal regions with a WELL-DEFINED THICK GREEN BAND FORMING A SEMI-CIRCLE at the tip of the sperm head (sperm #1). Score as acrosomal-reacted/abnormal acrosome if the acrosomal region is pink (sperm #2), or it is green but the semi-circle band is either broken-up, discontinuous, vesiculated, botched, missing band, or fuzzy, without a distinct dark-green band at the tip of sperm head (sperm #3).
15. Obtain the percent acrosome intact. Normal cut-off should be > 16 % sperm with normal intact acrosome. We obtained this number from the patient with the lowest percent acrosome intact that had fertilized oocytes in vitro.
    
    
16. Gently wipe off excess oil and store the stained slides in a dark drawer.

HELPFUL NOTES

1. Stain that become contaminated may be filtered prior to use.
2. Do not pour fixative or stains directly on to smear as this washes out some of the sperm.
3. It is feasible to pipet drop-by-drop the reagents on to the slide of sperm smear.
4. Use a tiny drop of oil for the analysis in order to preserve the other parts of the smear.
5. The use of Coplin jars to store the stain may result in evaporative loss of the stain or light-effects on the stain. It is best to keep the stain in the original plastic bottles and pipet out the drops as needed.

INTERPRETATION OF RESULTS

Acrosome intact sperm, "Green Head Sperm" -- The postacrosomal region and the nuclear portion of the sperm should stain RED. The acrosome, midpiece, and tail should stain GREEN. The sperm should show a normal head with even green staining of the intact acrosome with a semi-circle dark-green band at the tip of the sperm head. Since the acrosome is being analyzed rather than the morphology, all shapes of sperm that are "acrosome-intact" will be counted even though they appear as pyriform, amorphous, oval, etc.

Acrosome reacted sperm, "Red Sperm" -- The entire head region stains RED. This type of sperm is not desired because it has lost important sperm acrosome enzymes needed to penetrate the oocyte. Sperm cells with damaged head membranes, irregular staining, disruption or peeling of head acrosome should be counted as "Red Sperm." In our experience, we have found that globozoospermics "round-headed" sperm heads will be completely red in color.

The normal value has been tentatively identified as > 16 % of all sperm displaying acrosome-intact. When the percentage of acrosome-intact sperm falls below 16 %, then another analysis is performed to confirm acrosome problems. A grey zone exists between 16 % and 40 % acrosome intact where fertilization failure may occur. To interpret in the grey zone, other parameters (such as the percentage of strict normal morphology) must be taken into account.

To determine the percentage of acrosome reaction in response to a specific treatment as in THE ARIC TEST, or after a period of incubation, prepare the sperm smear before and after the treatment. Subtract the percent of sperm with intact acrosome after treatment from the percent intact acrosome before treatment. The difference should equal the percent acrosome reaction response. This is based on the assumption that the number of sperm with defective acrosome remains a constant. Remember that:

Total sperm = Intact acrosome + Reacted acrosome + Defective acrosome

CALCULATIONS

Analyze at least 100 sperm cells using the cell counter. Divide the number of green head sperm with green head band by the total number of sperm analyzed, multiplied by 100 to get the percent sperm with intact acrosome

OUT-OF-RANGE RESULTS

Acrosome-reacted sperm is but one of several semen analysis parameters that point to male factor infertility. If the percentage of acrosome-intact sperm (the green head sperm) is low, then fertility may be compromised. The other parameters such as sperm concentration, motility, hyperactivation, viability, as well as a repeat semen analysis on a different occasion must be considered before an accurate diagnosis can be made.

An extremely low percentage of acrosome-intact sperm (less than 4%) should be reported to the clinician or nurse. Enter the results into the computer and print the report for the clinician.

LIMITATIONS OF THE PROCEDURE

1. Note that with this stain, it is difficult to distinguish normally acrosome-reacted sperm from acrosome-defective sperm.
2. Good technique in preparing the sperm smears, such as eliminating thick seminal fluid should be used at all times otherwise the sperm will be trapped in a dark-green background.
3. The stain is only useful for sperm cells.

REFERENCES

1. Oettle EE: An improved staining technique which facilitates sequential monitoring of the acrosome state. Dev. Growth Differentiation (Suppl) 1986;28,96
2. Oettle, EE: Using a new acrosome stain to evaluate sperm morphology. Vet. Med. 1986,81:263
3. Chan PJ, Corselli JU, Jacobson JD, Patton WC, King A. Correlation between intact sperm acrosome assessed using the spermac stain and sperm fertilizing capacity. Arch. Androl. 1996;36:25-27.

Version: 9/96, P. Chan, Loma Linda University, Loma Linda, California, USA.