Sperm washing is important and how to do it

INTRODUCTION OF SPERM WASHING

• Artificial insemination is the first option treatment for infertile couples.
• Sperm washing is one technique used for artificial insemination required in such cases. Artificial insemination requires the selection of normal ejaculated spermatozoa before the performance of the treatment.
• It is necessary to remove other components of seminal fluid such as leucocytes, mucus and seminal plasma containing components such as prostaglandins, proteases, enzymes, mucoproteins, antibodies, and organic and inorganic components.
• Washed normal sperms are used for Intrauterine insemination (IUI) or in-vitro fertilization (IVF). Various types of sperm wash techniques are:
• 1) Basic sperm wash,
• 2) Density gradient centrifugation (premium wash)
• 3) Swim-up technique.

1. Importance
2. Requirement
3. choice of method
4. Basic sperm wash method
5. swim up method
6. Density gradient centrifugation
7. Procedure

Importance:-

• Leucocytes present in seminal fluid produce harmful reactive oxygen species. Similarly, various other components of seminal fluid (such as prostaglandins) may have adverse effects on fertilization after IUI or IVF.
• Hence, normal sperm are selected by sperm washing technique. Sperm washing is beneficial in the case of the following specific reasons.
• Unexplained infertility
• Male factor infertility
• Male with anti-sperm antibodies
• Women with endometriosis
• To decrease the risk of HIV transmission by HIV-positive fathers (since HIV infection is carried by the infected seminal fluid rather than sperm

Requirement:-

• Freshly collected semen in a sterile container After liquefaction, a “Routine semen examination” is performed.
• Biological safety cabinet.
• Seitz filters.
• Round bottom and conical centrifuge test tubes with a test tube stand.
• Centrifuge.
• Commercially available modified sperm washing medium (pH 7.2-7.4): It contains the following components: Sodium chloride, potassium chloride, magnesium sulfate, potassium phosphate, calcium chloride, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, phenol red and bovine albumin (5 mg/ml).
• Buffer solution: It contains 21 mm HEPES and 4 mm sodium bicarbonate
  After reconstitution in buffer solution, the sperm washing medium is filtered using a Seitz filter and aseptically processed. It is necessary to keep this medium at room temperature (25°C ± 5°C) for 30 minutes before use. Antibiotic supplementation may be added just before use.

choice of method:-

• For a semen sample with normal sperm count and motility, a basic wash or swim-up method is used.
• For a semen sample with poor sperm count and motility, the Density gradient centrifugation method is used.

Basic sperm wash method:-

1. Pipette of 2.0 ml liquefied semen aseptically around the bottom centrifuge tube
2. Add 2.0 ml sterile sperm washing medium and mix gently
3. Centrifuge at 300 g for 10 minutes
4. Remove supernatant and resuspend pellet in 1.0 ml sperm washing medium, mix gently
5. Centrifuge at 300 g for 5 minutes
6. Resuspend pellet in 1.0 ml sterile sperm washing medium
7. In the case of additional quantity of semen, repeat the steps 1-6
8. Perform sperm count on the washed sperm mixture and examine the motility of sperm. If satisfactory, use the sperm sample for IUI or IVF

swim up method:-

• By this method, the sperms are selected based on their rate of motility and capability to swim out of the seminal plasma.
• Pipette 1.0 ml liquefied semen aseptically in a found
• bottom centrifuge tube
• Add 1.3 ml sterile sperm washing medium and mix gently
• Place the tube in an inclined position at 45° and incubate at 37° for 30-60 minutes
• Place the tube in a vertical position and remove 1.0 ml supernatant aseptically
• In the case of the additional quantity of semen repeat steps 1-4
• Perform sperm count on the separated sperm mixture (supernatant fractions) and examine the motility of sperm. If satisfactory, use the sperm sample for IUI or IVF

Density gradient centrifugation:-

This method is used in the case of a semen sample with poor count and motility. By using a density discontinuous gradient, good quality sperms can be separated from semen plasma and other components of semen.
Additional Requirements:-
Commercially available ready-to-use “Stock density gradient medium” and “Isotonic sterile medium”. Antibiotic supplementation may be added just before use.
From the stock solution, the following two working solutions were prepared aseptically:
1 Working density gradient solution 1 (40% v/v): 4.0 ml of stock solution is mixed with 6.0 ml isotonic sterile
medium. Working density gradient solution 2 (80% v/v): 8.0 ml of stock solution is mixed with 2.0 ml isotonic sterile medium.

Procedure:-

• 1. Pipette 1.0 ml 40% (v/v) density gradient solution in a conical centrifuge tube
• 2. Add 1.0 ml 80% (v/v) density gradient solution 2
• 3. Add 1.0 ml liquefied semen
• 4. Centrifuge at 300g for 15 minutes
• 5. Remove supernatant and resuspend pellet in 5.0 ml sterile isotonic medium, mix g.ently and centrifuge at 200g for 10 minutes
• 6. Remove supernatant and resuspend pellet in 1.0 ml sterile sperm washing medium
• 7. In the case of the additional quantity of semen repeat steps 1-6
• 8. Perform sperm count on the final sperm mixture and examine the motility of sperm. If satisfactory, use the sperm sample for IUI or IVF