Antisperm AntibodyDetection Methods

by: Ahmad Ricardo

When an ASA assay should be obtained ?

1) the semen analysis shows sperm agglutination or clumping in the absence of clinical infection;

2) low sperm motility exists, with history of testis injury or surgery;

3) there is confirmation that increased round cells are leukocytes ;

4) sperm “shaking” is observed on sperm–cervical mucus contact testing;

5) poor penetration of mucus is observed on a postcoital test;

6) there is unexplained infertility.

Detection of Antisperm Antibodies

• Agglutination Tests

• Complement-Dependent Tests

• Immunoglobulin Binding Tests

• Mixed Antiglobulin Reaction and Immunobead Tests

• Enzyme Linked Immunosorbant Assays

• Other Tests

Agglutination Tests

• ability to cause agglutination

• observed macroscopically or microscopically

• tube agglutination test, gel agglutination test (Kirbrick), tray slide agglutination test (Franklin and Dukes)

• drawback:• agglutination can also occur in the

absence of specific antibodies as a result of the presence of bacteria, fungi, or amorphous material in seminal plasma, or under the influence of sex steroids or nonimmunoglobulin proteins in serum

Tube agglutination

test

Gelatin agglutination (Kirbrick) test

Microtray Agglutination Tests

• Friberg Test

• refference test

• titre, type, and degree of agglutination

• drawback:

• nonimmunological agglutination may occur

• particularly when female sera are tested, and at low serum dilutions

Complement-Dependent Tests (Isojima)• Induction of a cytotoxic

effect which occurs in the presence of complement

• Cytotoxicity results in the loss of sperm motility

• used as a marker

➢ the sperm immobilization test (SIT) or

➢ membrane damage ✓ need specific dyes

✓ ATP release cytotoxicity test (ARCT)→ objective

• disadvantage: can't detect IgA class antibodies

Immunoglobulin Binding Tests

• antiglobulins can either be labelled or bound to indicator cells or particles

• The antiglobulin label:➢ fluorescence molecule : immunofluorescence

technique

➢ radioactive isotope : radiolabelled antiglobulin assay

➢ an enzyme : enzyme-linked immunosorbent assay

• The antiglobulin indicators:➢ redblood cells : original mixed antiglobulin

reaction

➢ latex particles : Latex-MAR test

➢ polyacrylamide beads : immunobead binding technique

Mixed Antiglobulin Reaction Test

• presence of immunoglobulins bound to spermatozoa

• (red blood cells--immunoglobulins) + (motile spermatozoa--antiglobulin)

→ mixed agglutination

• the coated red blood cells have been replaced

→ coated latex particles

• MAR TEST

•direct : fresh semen

•indirect : blood serum, solubilized cervical mucus

Immunobead Tests

• polyacrylamide beads are coated with polyclonal antibodies against human IgG, IgA, or IgM

• requires washing the spermatozoa• repeated centrifugation-resuspension

• remove the bulk of immunoglobulins present in seminal plasma

• ≥ 50% or more adherence of particles to the motile spermatozoa : strong evidence for an immunological cause of infertility

• time consuming, less adequate, lower specificity and sensitivity than the SpermMAR test

Immunobead Tests

Enzyme Linked Immunosorbant Assays

• Antibody-enzyme-immunoglobulin complexes

• add a specific enzyme substrate → colourchange

• advantage : specific and quantitative

• disadvantage: the time and cost , poor sensitivity, and inability to determine ASA location and isotype.

Postcoital test (PCT)

• examining the cervical mucus several hours after intercourse for the presence of sperm

• prior to ovulation in the periovulatory phase

• a drop of cervical mucus is examined under wet-mount microscopy for the presence of sperm

• normal result :• ≥10–20 sperm per HPF (× 400)

• the majority : progressive motility

• abnormal: • sperm immobilized

• shaking motion in the cervical mucus (suspicious for the presence of antisperm antibodies)

Other Tests

• panning procedure for ASA detection on spermatozoa

• polyacrylamide gel electrophoresis and immunoblotting

• flowcytometry

Mixed Antiglobulin Reaction Test

• direct SpermMar Ig G Test method

• indirect SpermMar Ig G Test method

• direct SpermMar Ig A Test method

Direct SpermMar Ig G Test method

1. Allow the reagents and specimens to adjust to room temperature.

2. On a micro slide place:s➢ 10 microlitres of fresh untreated semen

➢ 10 microlitres of SpermMar IgG Latex Particle

➢ 10 microlitres of SpermMar IgG Antiserum

Direct SpermMar Ig G Test methodcon't

3. Mix the sample and the Latex reagent 5 times with the edge of a cover glass

4. Mix the Antiserum with the Latex reagent and sample mixture

5. The cover glass is put on the mixture and the mixture is observed under a light microscope using a 400x or a 600x magnification

6. Read the result after 2-3 minutes. Observe for latex particles attached to motile sperm. Count 100 spermatozoa to determine the percentage reactive sperm

7. If no attachement of beads to sperm is observed, read again after 10 minutes

Direct SpermMar Ig G Test method

Direct SpermMar Ig G Test method

Direct SpermMar Ig G Test method

Direct SpermMar Ig G Test method

Indirect SpermMar Ig G Test method

1. Allow all the reagents and specimens to adjust to room temperature.

2. Inactivate the serum specimens by heating them at 560 C for 30 minutes if glas test-tubes are used, 45 minutes if plastic test-tubes are used

3. Adjust the pH (by adding 0,1N NaOH or HCl) of the EBSS to 7,4-7,5

4. Wash the motile donor spermatozoa by letting them swim up at the pH adjusted medium (pH=7,4-7,5). Swim up has to be done in 5 ml glass or sterile plastic test-tubes with round bottom at 370 C for 45 minutes. Adjust the sperm concentration to 20x106 sp/ml with EBSS medium (pH= 7,4-7,5)

Indirect SpermMar Ig G Test methodcon't

5. Serially dilute the inactivated serum specimen 1/16 with EBSS medium (pH= 7,4-7,5) in a titre plate

6. Mix 50 microlitres of the (1/16) diluted, inactivated serum specimen (step 5) with 50 microlitres of the washed motile donor sperm donor sperm (step 4) in a free well on the titre plate. Incubate for 60 minutes at 37C.

7. On a micro slide place:➢10 microlitres of the sperm-serum mixture

➢10 microlitres of SpemMar IgG Latex Particle

➢10 microlitres of SpemMar IgG Antiserum

8. Mix the sample and the Latex reagent 5 times with the edge of a cover glass.

Indirect SpermMar Ig G Test methodcon't

9. Mix the Antiserum with the Latex reagent and sample mixture

10.The cover glass is put on the mixture and the mixture is observed under a light microscope using a 400x or 600x magnification (phase contrast or dark field illumination may also be used to fascilitate reading)

11.Read the results after 2-3 minutes. Observed for latex particles attached to motile sperm. Count 100 spermatozoa to determine the percentage reactive sperm. If no attachment of particle to sperm is observed, read again after 10 minutes.

Result• Test properly performed

• spem antibodies (+) → spermatozoa covered by latex particleand immobilized.

• Direct SpermMar IgG Test • Suspected : 10-39 %

• Highly probable : 40 %

• Result (+) → recommended to perform the SpermMar IgA test.

• Indirect Sperm IgG Test• Accepted : ≥ 40% reaction between the coated latex particle and motile

spermatozoa.

Inirect SpermMar Ig G Test method

Direct SpermMar Ig A Test method

1. Allow all the reagents and specimens to adjust to room temperature.

2. On a micro slide place:➢10 microlitres of the sperm-serum mixture

➢10 microlitres of SpemMar IgG Latex Particle

3. Mix the sample and the Latex reagent 5 times with the edge of a cover glass.

4. The cover glass is put on the mixture and the mixture is observed under a light microscope using a 400x or 600x magnification. The use of phase contrast or dark field illumination may also be used to fascilitate reading the slide.

Direct SpermMar Ig A Test method

5. Read the result after 2-3 minutes. Observe for latex particles attached to motile sperm. Count 100 spermatozoa to determine the percentage reactive sperm. Read again after 10 minutes.

6. The diagnosis• Suspected : 10-39 % attached to latex particle

• Highly probable : 40 % attached to latex particle

7. Occurrence of Mixed Aglutination Reaction (+):• ≥ 40 % in semen

• ≥ 10 % in cervical mucus

Research Data

• direct MAR test result for IgG correlated better with the indirect MAR result for IgG in seminal plasma and serum than did the direct immunobead test result for IgG

• the results of indirect MAR IgG test on serum correlate better than the immunobead test with the results of TAT and ARCT on serum

• Using the cut-off value of 40%, motile spermatozoa attached to coated latex particles as the lower limit of significant activity, the indirect MAR test has a sensitivity of 96% and specificity of 87% in comparison with the TAT test as a reference

Research Data

Refference

• Kompendium der Andrologie. From URL: http://www.med.uni-giessen.de/aka/andro/inhalt.html

• Mahmoud A, Comhaire F. Immunological Causes. Andrology for the Clinician. Springer-Verlag Berlin Heidelberg 2006. 47-51

• Mahmoud A, Comhaire. F. Semen analysis What’s new. Presentation from URL:http://slideplayer.com/slide/7283211/

• Ong F. Semen Analysis and Sperm Preparation. Presentation from URL:http://slideplayer.com/slide/4687961/

• Pourmand G. Medical Therapy in Male Infertility. 9 th Royan Int. Congress. Presentationfrom URL: http://www.slideshare.net/patriciakh/infertility2-1923189

• Sperm Antibody Testing. From URL: http://fertilitysolutions.com.au/sperm-antibody-testing/

• SpermMar™ /MAR-Test (mixed antiglobulin reaction) . IVF Express. Presentation from URL:https://www.ivf.express/products/1392?hl=en

• Ulcova-Gallova Z. Reproductive Immunology. Presentation from URL:http://slideplayer.com/slide/10413210/

• Walsh TJ, Turek PJ. Immunologic infertility. Infertility in the Male Fourth Edition. CambridgeUniversity Press. 2009 (16):277-89

•Thank You