This document discusses various sperm preparation methods used for intrauterine insemination (IUI). It describes the simple wash method, swim-up method, and density gradient centrifugation method. For each method, it provides the steps, including centrifugation speeds and times. It also covers preparing samples for retrograde ejaculation and samples from HIV-infected patients. The goal of sperm preparation is to select motile sperm and remove other seminal constituents like debris to maximize the chances of fertilization during IUI.

1. IUI-Sperm Preparation
Methods
DR.RENUKADEVI
SENIOR EMBRYOLOGIST
ARC International Fertility & Research
Centre

2. Steps of IUI
 Pretreatment workup
 Counselling for IUI
 Ovarian Stimulation
 Monitoring of Response
 Ovulation Trigger & Timing of IUI
 Semen Collection
 Sperm Preparation
 Insemination
 Post Insemination follow up

3. What is IUI
 IUI is deliberate introduction of sperms in the female uterus in
order to achieve pregnancy
 One of the simplest techniques of ARTs
 Minimally invasive and uncomplicated procedure
 First therapeutic step in treatment of infertility
 Cost –effective, simple, can be learned easily
 Husband or Donor sperm (frozen and quarantined) can be used.

16. Sperm Preparation Methods
• Semen is a mixture of motile, non-motile and dead
spermatozoa with cells, cellular debris and sometimes micro-
organisms.
• There are various sperm separation and isolation methods.
• These methods try to replicate in vitro the natural process in
which viable sperm are separated from other constituents of
the ejaculate as they actively migrate through the cervical
mucus.

17. • The World Health Organization recommendation is to
process sperm sample within one hour after ejaculation in
order to prevent damage from leukocytes and other cells
present in the semen.
• The sperm suspension obtained using these methods is used
for IUI, IVF, ICSI and different sperm tests.

18. Intrauterine Insemination
(IUI)
Goal is to Maximize the Chance of Fertilization
• Increase Number of Eggs
• Position Sperm Closer to Eggs

19. The most commonly used sperm
preparation methods in ART are :
Simple wash
• This method is used if the semen sample is very poor and it
mainly removes seminal plasma from the sperms. This
procedure provides the highest yield of spermatozoa.
• Therefore it is often used for IUI.
• In this procedure the culture medium is added to the ejaculate
after liquefaction and the mixture is centrifuged twice using
lower centrifugation forces and fewer centrifugation steps in
order to minimize the damage caused by ROS.

20.  Mix the semen sample well
 Dilute the entire semen sample 1 + 1 (1:2) with supplemented medium to promote removal of
seminal plasma.
 Transfer the diluted suspension into multiple centrifuge tubes, with preferably not more than 3 ml
per tube.
 Centrifuge at 1200g for 5–10 minutes.
 Carefully aspirate and discard the supernatants.
 Resuspend the combined sperm pellets in 1 ml of supplemented medium by gentle pipetting.
 Centrifuge again at 1200g for 3–5 minutes.
 Carefully aspirate and discard the supernatant.
 Resuspend the sperm pellet, by gentle pipetting, in a volume of supplemented medium appropriate
for final disposition, e.g. insemination, so that concentration and motility can be determined (Who
laboratory manual for the examination and processing of human semen 2010)
The Common Steps of this Method are:

21. SWIM-UP
 This method is used for semen samples with normal
count and motility.
 Allow the semen sample for liquefaction
 Microscopic assessment of the sample should be done
to confirm count and motility.
 Label the conical tubes with the patients name & ID.

22. Swim Up Method
This technique relies on the ability of spermatozoa to swim out of seminal plasma
into culture medium and is suitable for semen with high to moderate motility. This
procedure selects spermatozoa for their motility.
The common steps of this method are:
 Evaluate the percentage of motile and total number of sperm on a Makler
Chamber
 Fill the round-bottom test-tubes with 2 ml Sperm Preparation Medium (medium)
 layer 1ml of the liquefied semen underneath the medium

23.  Incubate the sample in the incubator for 60 min. Placement of tube at 45°
angle creates a larger surface area for sperms to swim-up.
 Aspirate the medium of the upper part of the test-tubes into new prewar med
round-bottom test-tubes without disturbing the lower layer
 Add 5 ml of the medium to the sample and centrifuge 5 min/1200 rpm
 Aspirate the supernatant but save 0.5 ml of the medium to resuspend the
pellet.
 Evaluate the percentage of motile and total number of sperm on a Makler
Chamber

24. Swim Up Method wash media (2ml)
Semen sample
***Semen sample, wash media vol must
be equal
Centrifuge normal sample 10 mins 1200 rpm.
Viscous Sample 15mins 1500 rpm
Discard Sup.
Add wash media (2ml) & mix gently sample
Centrifuge Sample 5 mins 1200 rpm
Discard Supernatant
Add 1 ml of Wash Media w/o Directly
Pouring it on the Pellet
(Around the Sides of the Tube)
Incubation: Tube Slanted
for 30 mins at 45o.
All Motile Sperms
Swim up to the Surface.
After Incubation Sup. Taken out
and given to Doctor, pellet
Discarded

25. DENSITY GRADIENT
This method is used to wash poor quality of semen
samples.
 Low motility
 Poor forward progression
 Large amount of debris
 High number of cells
 Antisperm Antibodies
 Highly viscous

26. Density-Gradient Systems
 Density gradient centrifugation separates cells based on their density.
 Morphologically normal and abnormal spermatozoa have different densities
and at the end of centrifugation they are located accordingly.
 The solutions like colloidal silica, poly-sucrose and others have higher
density than semen. Because of that this system can separate the debris, cells,
microorganisms and non-motile sperms from the motile ones.
 Centrifugal force enables the motile sperms to swim from a less dense
seminal fluid into a denser solution. Cellular debris and non-motile
microorganisms are trapped at the interphase between the two solutions.

27. High Density Gradient (80%)
Low Density Gradient (40%)
Semen Sample
Centrifuge 2000 rpm 10 – 15 mins; 15 – 20 mins for high viscous
2 ml wash media poured into new tube, then pellet mixed gently into new tube
Sup discarded
Centrifuge 1200 rpm 5 mins
Remove sup., add 0.5 ml wash media to the pellet, then mix well
Load the sample for analysis
DENSITY GRADIENT TECHNIQUE
*** Semen Sample, gradient
volumes must be equal

29.  Evaluate the precentage of motile and total number of sperm on a
Makler Chamber
 Layer 2 ml of the lower density medium (40% ) into conical testtube
 Put the layer of the high density medium (80%) underneath the upper
layer (lower layer will raise and a sharp interface is formed)
 Layer 2 ml of the liquefied semen on the top of the gradient
 Centrifuge the sample 15 min/1200 rpm
 Use a new sterile Pasteur pipette to aspirate, in a circular movement
from the surface, everything except the pellet and 4-6 mm of lower
phase.
The common steps of this method are:

30.  If no pellet is seen after centrifugation, remove all fluid except the lowest
0.5ml.
 Aspirate the pellet or the lowest 0.5ml liquid. Transfer sperm pellet to
new tube and re-suspend pellet in 5ml sperm wash.

31.  Centrifuge the sample at 1200 RPM for 5 minutes. Do not use the brake.
 Aspirate sperm wash supernatant leaving as little (0.25-0.50ml according to the sperm
count and motility) liquid as possible above pellet for swim-up.
 Add 1.5ml of media without disturbing the pellet.
 Incubate the tubes at a 45° angle for 20 – 30 minutes for swim-up in vertical rack in a
37°C incubator.
 Aspirate sperm wash supernatant above pellet and mixed with suitable volume of culture
medium to obtain the required sperm concentration.
 Evaluate the precentage of motile and total number of sperm on a Makler Chamber

32. Preparing Retrograde
Ejaculation Samples
 In some men, semen passes into the bladder at ejaculation,
resulting in aspermia, or no apparent ejaculate.
 Confirmation of this situation is obtained by examining a
sample of post-ejaculatory urine for the presence of
spermatozoa.
 If pharmacological treatment is not possible or not
successful, spermatozoa may be retrieved from the urine.

33.  Alkalinization of the urine by ingestion of sodium
bicarbonate, for example, will increase the chance that any
spermatozoa passing into the urine will retain their motility
characteristics.

34.  At the laboratory, the man should be asked to:
 Urinate without completely emptying the bladder;
 Produce an ejaculate by masturbation into a specimen container.
 Urinate again into a second specimen vessel containing culture medium
(to alkalinize the urine further).
 Both the ejaculate, if any, and urine samples should be analysed.
 Because a large volume of urine may be produced, it is often necessary
to concentrate the specimen by centrifugation (1200g for 5-10 minutes).
 The retrograde specimen, once concentrated, and the antegrade
specimen, if produced, can be most effectively processed using the
density-gradient preparation method.

35. Preparing HIV-infected
semen samples
 If the human immunodefciency virus (HIV) is present in
semen, viral RNA and proviral DNA can be found free in
seminal plasma and in non-sperm cells.
 As HIV receptors (CD4, CCR5, CXCR4) are expressed only
by non-sperm cells, a combination of density-gradient
centrifugation followed by swim-up has been proposed as a
way of preventing infection of uninfected female partners.

36. References
 WHO Laboratory manual for the examination and processing of
human semen. Fifth edition, 287 pages, ISBN 9789241547789,
prepublication version, 2010
 A. Aribarg, N. Sukcharoen, 1995 intrauterine insemination of washed
spermatozoa for treatment of oligozoospermia. Int J Androl, 18 1
(1995), 62 66

37.  Textbook of Assisted Reproductive Techniques- Laboratory
and Clinical Perspectives by David K Gardner, Ariel
Weissman Colin M Howels, Zeev Shoham, 2nd Edition
 Textbook of In Vitro Fertilization and Assisted
Reproduction- The Bourn Hall Guide to Clinical and
Laboratory Practice, 3rd Edition, Edited by Peter R Brinsden

38. THANK YOU