Professional Diploma in Clinical Embryology

Sperm preparation

Separate sperms from seminal plasma to

yield a final preparation containing a
high percentage of morphologically
normal and motile sperms, free from
cells-debris, cells, non-germ cells and
dead sperms.
 Sperm preparation cont …
The choice of sperm preparation method or combination
of methods depends on:

 Volume
 Sperm count.
 Sperm morphology.
 Motile count.
 Ratio between motile : immotile counts
 Presence of antibodies, agglutination, pus cells or debris.
 Methods
1. Washing or concentration.

2. Swim-up

3. Double swim-up

4. Discontinuous density gradients

 Washing or concentration
Provides the highest yield of sperms.

Useful for viscous samples, very low sample volume,

motility is very poor and the total concentration of semen
is very low.

 Mix the semen sample well.

 Dilute the semen sample 1 + 1 (1:2) with media to
remove seminal plasma.
 Transfer the diluted suspension into centrifuge tube.
 Washing or concentration cont …
 Centrifuge at 300–500g for 5–10 minutes.
 Carefully aspirate and discard the supernatants.
 Re-suspend the pellets in 1 ml of media by gentle
pipetting.
 Centrifuge again at 300–500g for 3–5 minutes.
 Carefully aspirate and discard the supernatant.
 Re-suspend the pellet, by gentle pipetting, in a
volume of media appropriate for final disposition.
INSEMINATION- SPERM PREPARATION.webm
 Swim-up or layering
 Mix the semen sample well.
 Place 2.0 ml of semen in a sterile 15-ml conical
centrifuge tube, and gently layer 1.2 ml of media
over it.
 Put the tube at an angle of about 45°, to increase
the surface area, and incubate for 1 hour at 37
°C.
 Gently return the tube to the upright position.
 Swim-up or layering cont …
 Aspirate uppermost 1 ml of media to new tube,
(contain highly motile sperm cells).
 Add 1.5–2.0 ml of media.
 Centrifuge at 300–500g for 5 minutes and discard
the supernatant.

 Re-suspend the sperm pellet in 0.5 ml media for

assessment of sperm concentration, total motility
and progressive motility, or carefully add 1.0 ml
media for double swim up.
Sperm Preperation For IUI_HD.mp4
 Pellet and swim-up
This method is not recommended when:

1. motility is poor.

2. Large amount of cellular contamination and

debris.

3. Viscous samples
 Pellet and swim-up cont …
 Mix the semen sample well.

 Dilute the entire semen sample 1 + 1 (1:2) with

media to remove seminal plasma.

 Transfer the diluted suspension into centrifuge

tube.

 Centrifuge at 300–500g for 5–10 minutes.

 Carefully aspirate and discard the supernatants.
 Pellet and swim-up cont …
 Carefully add about 0.75 ml (or 1 ml) of media over
the pellet, taking care not to disturb it.
 Put the tube at an angle of about 45°, to increase
the surface area.
 Allow the sperm to swim up into the media.
 Carefully remove supernatant from pellet and place
in a clean centrifuge tube.
 Pellet and swim-up cont …
 Centrifuge again, re-suspend in 1.0 ml media to
assess count and motility or carefully add 1.0 ml
media for double swim up.

10 minutes is sufficient for very motile sperm

1 hour plus may be required for poorly motile

sperm.
2 iui sample preparation swim up method.webm
 Discontinuous density gradient
Discontinuous density gradients can provide the best
selection of good-quality sperm, giving good
separation from other cell types and debris.

Decreased the yield.

Debris, round cells, and abnormal forms with

amorphous heads and cytoplasmic droplets never
reach the bottom of the tube because of their low
density.
 Two-step gradient (80/40)
Can be used for all samples which contain > 4×10^6
motile sperm/mL .

Specimens with known or suspected anti-sperm

antibodies.

80%: 8 mL isotonic + 2 mL culture medium.

40%: 4 mL isotonic + 6 mL culture medium.

 Two-step gradient (80/40) cont…
• Gradients: pipette 2.0–2.5 mL of 80% into the
bottom of a conical centrifuge tube, and gently
overlay with an equal volume of 40%.

 Layer up to 2 mL of sample on top of the 40% layer.

 Centrifuge at 600 g for 20 minutes.

 Carefully recover the pellet at the bottom of the 80%

layer, re-suspend in 1 mL of media.
 Two-step gradient (80/40) cont…
 If the sample looks sufficiently clean, centrifuge for
5 minutes at 200 g , re-suspend the pellet in fresh
media, and assess the final preparation.
 If there is a high percentage of immotile sperm,
centrifuge at 200 g for 5 minutes, remove the
supernatant, carefully layer 1 mL fresh medium
over the pellet, and allow the motile sperm to swim
up for 15–30 minutes.
 Collect the supernatant and assess the final
preparation.
 Mini-gradient (95/70/50)
95%: 9.5 mL gradient solution + 0.5 mL culture
medium
70%: 7.0 mL + 3.0 mL

50%: 5.0 mL + 5.0 mL

 Gradients: make layers with 0.3 mL of each solution:

95, then 70, then 50.
 Mini-gradient (95/70/50) cont …
 Dilute the semen 1:1 with culture media.

 centrifuge at 200 g for 10 minutes .

 Re-suspend the pellet in 0.3 mL culture medium,

and layer over mini-gradient.

 Centrifuge at 600 g for 20–30 minutes.

 … Mini-gradient (95/70/50) cont
 Recover the pellet, re-suspend in 0.5 ml culture
media, and assess count and motility.

 Proceed exactly as for two-step gradient

preparation: either centrifuge at 200 g for 5
minutes and re-suspend the pellet, or layer over
the pellet for a further preparation by swim-up.
 Enhancement of sperm motility
for insemination
Pentoxifylline increase in intra cellular adenosine
triphosphate (ATP) enhances sperm motility.

30-minute preincubation of prepared sperm with the

stimulant.

 The resulting sperm suspension is then washed to

remove the stimulant.

Prepare immediately for used.

Thank You