Ms. Doel Bose Pande discusses various semen preparation methods and principles. Common techniques include simple wash, swim-up, and density gradient centrifugation. The choice of method depends on factors like semen quality, intended use, and practical considerations. Density gradient is best for separating motile sperm from debris but is more time-consuming than direct swim-up. The goal is to recover high-quality sperm with minimal processing time and damage. Practical issues like sample volume and number of patients may also influence the choice of preparation technique.

1. Ms. Doel Bose Pande
Embryologist & Clinic Manager
@
Indore Infertility Clinic, Indore
Contact: 96444 44066
Semen Preparation Methods
Principles & Techniques

2. Content
• Purpose of Sperm Separation
• Common Techniques
 Simple Wash
 Swim-up
 Density Gradient
• Factors Determining the choice of method
• Efficiency of Sperm Selection Technique
• Preparing TESA/PESA Samples
• Take Home Message

3. Facts
• The spermatozoa of all placental (eutherian) mammals, including
humans, are in a protective, nonliable state at ejaculation and are
incapable or poorly capable of fertilization even if they are placed in
direct contact with an oocyte.
• They must undergo a subsequent period of final maturation
(Capacitation) during which they acquire the capacity to interact
with the oocyte–cumulus complex and achieve fertilization.

4. Why Sperm Separation ?
• Seminal plasma contain one or more factors (e.g. prostaglandins, zinc, leukocytes)
that prevent spontaneous capacitation of spermatozoa upon ejaculation
• Prolonged exposure to seminal plasma has adverse effects on sperm function :
– Like the ability to penetrate cervical mucus (Kremer, 1968)
– undergo the acrosome reaction in vitro, and the fertilization process in general
(Rogers et al, 1983; Mortimer and Mortimer, 1992; Mortimer et al, 1998).
• Consequently, in order for a spermatozoa to have the capacity to fertilize an
oocyte, they must be separated from the seminal plasma.

5. Why Sperm Separation ?
• The World Health Organization recommendation is to process
sperm sample within one hour after ejaculation in order to
prevent permanent damage from leukocytes and other cells
present in the semen.
• Recommended Time – 20 to 30 mins

6. Purpose of Sperm Preparation
• To yield a final preparation that has high percentage of sperms that are:
 Motile
 Morphologically Normal
 Free from debris, non-germ cell and dead sperms
• Efficiency of any sperm preparation method is expressed in terms of :
 Absolute Sperm Numbers
 Number of Motile Sperms
 Recovery of Morphologically normal Motile Sperms

7. Common Techniques
Principles
Common Techniques routinely used

8. Common Techniques
• There are 4 basic approaches to sperm preparation:
 Washing (e.g Simple Wash)
 Sperm Migration (Migration of healthy sperm based on self propelled movement. e.g. Swim-Up)
 Adherence
 Filtration
Density Gradients (Combination of the sperms’ own motility and their adherence to filtration
matrices and the retention at phase borders)
Adherence methods to eliminate debris and dead spermatozoa (e.g. Glass Wool, Glass beads, and
Sephadex columns)

9. Common Techniques
Simple Wash

10. Simple Wash
• Provides the highest yield of spermatozoa and is adequate if
semen sample is of optimal quality.
• It is often used for preparing IUI samples from cryopreserved
semen.

11. Simple Wash - Method
1) Mix the semen sample well
2) Dilute the entire semen sample 1 + 2
(1:2) with supplemented medium to
promote removal of seminal plasma.
1) Transfer the diluted suspension into multiple centrifuge tubes, with
preferably not more than 3 ml per tube.
2) Centrifuge at 300–500g for 5–10 minutes.

12. Simple Wash - Method
5) Carefully aspirate and discard the
supernatant.
5) Resuspend the combined sperm pellets in
1 ml of supplemented medium by
gentle pipetting.
5) Centrifuge again at 300–500g for 3–5 minutes.
6) Carefully aspirate and discard the supernatant.
7) Resuspend the sperm pellet, by gentle pipetting, in a volume of
supplemented medium appropriate for final insemination

13. Common Techniques
Swim - Up

14. Swim-Up
• It is a migration-based technique.
• Swim-up is one of the most commonly used techniques for
sperm preparation.
• In Conventional swim-up, a pre-washed sperm pellet obtained
by a soft spin is placed in an overlaying culture medium in a
conical tube. So it involves swim-up from the cell pellet rather
than direct semen.

15. Traditional Swim-Up

16. Direct Swim Up
Direct swim-up (without diluting semen) is the simplest and fastest method for
separating sperm by migration for Oligospermic samples.

17. Direct Swim Up - Method
• Allow specimen to completely liquefy for 15 to 30 mins
• Place 1 ml of semen in a sterile 15-ml round bottom
tube, and gently layer 2 ml of medium
over it.
• Incline the tube at an angle of about 45°, to increase the
surface area of the semen–culture medium interface,
and incubate for 30 to 60 minutes at 37 °C.
• Gently return the tube to the upright position and remove the uppermost appx 1.5
ml of medium. This will contain highly motile sperm cells.

18. Direct Swim Up - Method
• Dilute this with 1.5–2.0 ml of medium.
• Centrifuge at 300–500g for 5 minutes & discard the supernatant.
• Resuspend the sperm pellet in 0.5 ml of medium
Note:
• To increase number of motile sperm in final inseminate, use multiple tubes with small
volume which will further increase the interface area.
• Do not use unsupplemented medium as it might result in sperm sticking to the surface of the
test tube leading to a lower yield and motility.

19. Disadvantages of Swim Up
• The swim-up method is simple and relatively inexpensive, yet it has some
disadvantages:
 Centrifugation performed in conventional swim-up has been shown to generate ROS.
 Many layers of the sperm pellet may cause potentially motile spermatozoa in the lower
levels of the pellet never to reach the interface with the culture medium layer.
 Amount of motile spermatozoa retrieved is relatively low (< 20% of motile sperms
retrieved)
 Takes twice as long as some other routinely performed techniques.

20. Common Techniques
Density Gradient

21. Density Gradient - Principle
• Density gradient centrifugation separates sperm cells based on their density. Thus,
at the end of centrifugation, each spermatozoa is located at the gradient level that
matches its density.
• Morphologically normal & abnormal sperms have different densities:
– Normal Mature Sperm – at least 1.10 g/mL
– Abnormal Immature Sperm – Between 1.06 – 1.09 g/mL
• As a result the leukocytes, cell debris and morphologically abnormal and poor
motility sperms are trapped inside the interphases.

22. Density Gradient - Principle
• Density gradients can either be continuous or discontinuous.
 Continuous Density Gradient - There is a gradual increase in density from the
top of the gradient to its bottom.
 Discontinuous Density Gradient - There are clear boundaries between lower
and upper gradients. The most widely used discontinuous density gradient
preparation includes 45% (v/v) density (top layer) and 90% (v/v) density (lower
layer).
• Double Density Gradient is the most standard and most widely used density
gradient preparation.

23. Density Gradient - Principles
• Components of the density gradient sperm separation procedure include a
colloidal suspension of silica particles stabilized with covalently bonded
hydrophilic silane supplied in HEPES.
• There are two Gradients:
– Lower Phase (90%)
– Upper Phase (45%)
• Sperm washing medium is used to wash the resuspended final pellet.

24. Discontinuous Density Gradient
• Discontinuous density-gradients can provide the best selection of good-quality
spermatozoa, giving good separation from other cell types and debris.
• It is easy to perform and yields higher sperm concentrations with less preparation
time (approximately 55 minutes)
• It is easier to standardize than the swim-up technique, and thus results are more
consistent.
• This technique is used to recover and prepare spermatozoa for use in IUI, IVF and
ICSI.

25. Density Gradient

26. Discontinuous Density Gradient
• Place all components of the upper and lower phase, sperm wash
and semen sample in incubator at 37°C for 20 mins
• Transfer 1mL of the lower phase into sterile conical bottom tube.
• Layer 1 mL of the upper phase on top of the
lower phase very slowly without disturbing the
lower phase using a transfer pipette.
• A distinct line separating the two layers called ‘Interphase’ will be observed.
This two-layer gradient is stable for up to 1 hour.

27. Discontinuous Density Gradient
• Gently place 1 ml of liquefied well mixed semen sample over the layered gradient.
You may use more than one tube based on the count, motility and quantity of
semen sample to generate a better yield.
• Centrifuge for 12 to 15 mins at 300g.
• Remove most of the supernatant from
the sperm pellet.
• Resuspend the sperm pellet in 2 to 3 ml of
supplemented medium by gentle pipetting and
centrifuge at 200g for 4–10 minutes.

28. Discontinuous Density Gradient
• Remove most of the supernatant
from the sperm pellet
• Resuspend the final pellet in
supplemented medium by gentle
pipetting so that concentration and
motility can be determined

29. Tips for maximizing Yield
• Avoid overloading the gradient with semen sample as it causes a
phenomenon called ‘rafting’’ i.e aggregation of undesirable
components of semen in the post centrifugation pellet.
• Use the gradient within one hour after layering it as eventually the
two phases will blend over time and sharp interface will cease to
exist.
• A three gradient column (40% -70% - 90%) can be used to remove
the cryopreservatives from thawed semen samples.

30. Revisiting the Methods

31. Not so Common Methods
 Test-Yolk Buffer
 Migration Sedimentation using Tea-Jondet Tubes
 PVP Droplet swim out for ICSI
 Glass-wool Filtration, Glass Bead Filteration
 Electrophoresis and Flourescence Cell Sorting
 Swim-Down
 Sephadex Column
 Transmembrane Migration

32. Factors Affecting Choice of Method
What Factors determine the choice of
method used for Sperm preparation?

33. Choice of Sperm Preparation Method
• The ideal sperm separation technique should :
 be quick, easy and cost-effective
 isolate as much motile spermatozoa as possible
 not cause sperm damage or non-physiological alterations of the separated
sperm cells
 eliminate dead spermatozoa and other cells, including leukocytes and bacteria
 eliminate toxic or bioactive substances like decapacitation factors or reactive
oxygen species (ROS)
 allow processing of larger volumes of ejaculates
• None of the methods available meets all these requirements

34. Comparison
Technique Used Time Cost Yield Quality
Simple Wash Low Low High Low
Direct Swim Up Highest Low Lowest Highest
Density Gradient High Highest Highest High
*Comparison made for the same volume of liquefied sample prepared.

35. Choice of Sperm Preparation Method
• Depends on nature of semen sample
• Direct Swim Up - Normozoospermic
• Double Density Gradient – Oligozoospermic,
Teratozoospermic, Asthenozoospermic

36. Preparing PESA/TESA Samples
PESA/TESA Samples

37. Preparing PESA Samples
• Sperm samples obtained from the epididymis do not contain a
significant amount of non-germ cells such as red blood cells.
• If sufficient numbers of epididymal sperm cells are collected,
density gradient centrifugation can be used to prepare the
spermatozoa for ART.
• If number of spermatozoa aspirated is low, simple wash
technique can be used.

38. Preparing TESA Samples
• Testicular samples contain large numbers of non-germ cells such as red
blood cells. Spermatozoa need to be separated from these non-germ cells.
• To free the testicular spermatozoa from the seminiferous tubules, use
mechanical (Teasing/Mincing) or enzymatic (Collagenase) method.
• Erythrocyte-lysing buffer (ELB) can be used to remove RBC.
• Pentoxyfylline is occasionally used to increase the sperm motility.

39. Preparing TESA Sample
• Use Simple Wash to prepare the specimen obtained by adding 1.5 ml of culture
medium.
• Centrifuge at 300g for 8–10 minutes.
• Remove the supernatant and resuspend the pellet in 0.5 ml of fresh culture
medium.
• Estimate the motility and number of spermatozoa in the pellet.
• (Some specimens with a low number of spermatozoa may need to be resuspended
in a lower volume of medium.)

40. Treating SHV (Semen Hyper Viscosity)
• Viscous semen samples can be treated with following enzymes and
incubated further in order to achieve liquefaction:
Proteolytic Enzyme
Bromelain
Trypsin
N-Acetyl Cysteine (NAC)
• Please follow manufacturers recommendation for processing
viscous semen sample with the enzyme of your choice and process
semen using Density Gradient.

41. Practical Factors
• Practical Factors affecting our choice:
When multiple IUI/IVF/ICSI Sample needs to be
prepared at the same time. How many samples should
you process at the most? Preference to which sample?
Processing Oligospermic sample high in volume ?
Handling sample with high concentration of motile
sperms

42. Take Home Message
• Centrifugal pelleting of unselected populations of human spermatozoa
causes irreversible damage to the spermatozoa that can impair—even
totally destroy—their fertilizing ability
• The method of choice is dependent on the intended ‘use’ of the prepared
sample. e.g. IUI needs a much higher yield as compared to ICSI
• Many practical scenario on day to day basis may affect the choice of
semen preparation method, use your learning wisely to choose the
appropriate method

43. Thank You