SEMEN PREPARATION TECHNIQUES FOR IVF IN EMBRYOLOGY

SEMEN PREPARATION
SWIM-UP
DENSITY GRADIENT
ADVANCED SEMEN PREPARATION METHODS

1. SEMEN PREPARATION
Dr. Deepthi Repalle Ph.D.
IVF Lab Director
MOHAK IVF

2. IMPORTANCE
The aim of sperm preparation for the ART is to
maximize the chances of fertilization
Sperm characters important for fertilization are
normal morphology, normal intact acrosomes,
progressive motility, ability to bind to zona
pellucida, penetrate the zona pellucida, fuse
the oolemma and form a male pronucleus

3. SEMEN PREPARATION-why?
To remove the seminal plasma which has decapacitation factors
Protection of sperm from non sperm cells
Free of debris, germinal cells, dead sperms
Improves sperm motility, morphology, survival and preserves the DNA
damage by ROS
Removal of contaminants
Osmolality is maintained

4. SEMEN PREPARATION-when?
Semen sample must be prepared prior to being used for IUI, IVF and ICSI
techniques.
Before semen cryopreservation/ fertility preservation
Generally the semen samples should be prepared within one hour of
ejaculation to prevent the detrimental effects
In presence of antibodies.

5. SEMEN PREPARATION-where?
The sperm preparation should be performed in a clean sterile work area
preferably.
Non-toxic, non-powdered gloves should be worn while handling semen
sample.
Sample should be collected in sterile
non-toxic semen collection container.
Semen sample should be collected not more than
1 hr. before preparation.
A thorough identification protocol of the patient should be performed.

6. SEMEN PREPARATION-how?
Different methods are there for sperm preparation
Choice of the method-nature of the sample
If the sperm count and motility are adequate, simple
washing and swim up is suitable.
If the semen quality is poor and includes large number of
other cells, gradient centrifugation is preferred

7. SEMEN PREPARATION-how?
The sperm should be prepared soon after
liquefaction of the seminal plasma
The sample must be mixed thoroughly
because ejaculation does not result in a
homogeneous suspension of sperm in seminal
plasma
The medium chosen should be equilibrated
with the gas mixture (bicarbonate buffer) and
the temperature maintained constant at 37◦c
(HEPES or MOPS).

8. Types of medium used for
preparation
 Sperm preparation medium(HEPES/MOPS)
 Density gradient medium (silane coated
colloidal silica particles suspended in HEPES)
 IVF medium(Bicarbonate)

9. Sperm preparation media
COMPOSITION:
Synthetic serum replacement
Human serum albumin
Sodium pyruvate
Sodium Bicarbonate
Penicillin
Streptomycin
HEPES/MOPS
Phenol red

10. High molecular weight fractions from serum
appear to be important for sperm motility.
Antibiotics reduce the risk of transmitting
infections
Bicarbonate ions are required for capacitation of
sperm.
Sperm preparation media

11. SEMEN PREPARATION METHODS
Simple washing
Direct Swim -up
Discontinuous Density Gradient

12. SIMPLE WASHING
Mix the semen sample well
Semen sample and media are mixed in 1: 2 Ratio to promote
the removal of seminal plasma.
Centrifuge at 300-500g (1500-1800 rpm) for 5-10 minutes
Carefully aspirate and discard the supernatants
Resuspend the combined sperm pellets in 1 ml of medium
by gentle pipetting.
Centrifuge again at 300-500g for 3-5 minutes.
Carefully aspirate and discard the supernatant.
Resuspend the sperm pellet, by gentle pipetting in a volume
of medium appropriate for final disposition

13. SIMPLE WASHING
pellet pellet
semen
media
1
st
spin 2
nd
spin
Resuspend
the pellet
Discard the
supernatant
Discard the
supernatant
Media
+
pellet
Resuspend
the pellet
Media
+
pellet

14. Advantages Disadvantages
Easy to perform
Very cost effective
Recover very clean fraction of
highly motile spermatozoa
Restricted to ejaculate with
high sperm count and motility
Sperms can be damaged by
ROS
Contamination remains
Do not eliminate debris and
leukocytes

15. Swim-up
Similar to the washing procedure
The centrifuged tubes are incubated at an angle 45
o
c.
For an incubation period of 30-45 minutes, following incubation, the
top portion of the overlay is used for the desired insemination
method.
Swim-ups are unsuitable for oligo and asthenozoospermic samples-
they yield very low number of motile spermatozoa.
Highly viscous samplesalso respond poorly to the swim-up
technique.

16. Direct swim-up
The direct swim-up can be performed by layering culture
medium over the liquefied semen.
Motile spermatozoa swim into the culture medium.
This procedure gives a lower yield of spermatozoa than
washing but selects them for their motility and is useful
where the percentage of motile spermatozoa in semen is
low e.g. IVF AND ICSI
Ideal for normozoospermic samples

17. DIRECT SWIM-UP METHOD
PROCEDURE:
Mixthesemensamplewell.
Place1mlofsemeninasterile15-mlconicalcentrifugetube,andgentlylayer1.2
mlofmediumoverit.Alternatively,pipettethesemencarefullyundertheculture
medium.
Inclinethetubeatanangleofabout45°,toincreasethesurfaceareaofthesemen–
culturemediuminterfaceandincubatefor1hourat37°C.
Gentlyreturnthetubetotheuprightpositionandremovetheuppermost1mlof
medium.Thiswillcontainhighlymotilespermcells.
Dilutethiswith1.5–2.0mlofmedium.
Centrifugeat300–500gfor5minutesanddiscardthesupernatant.
Resuspendthespermpelletin0.5mlofmediumforassessmentofsperm
concentration,totalmotilityandprogressivemotility.
Thespecimenmaybeuseddirectlyfortherapeuticorresearchpurposes

18. DIRECT SWIM-UP METHOD
media
45° 1ml

19. Advantages Disadvantages
Easy to perform
Very cost effective
Recover very clean fraction of
highly motile spermatozoa
Restricted to ejaculate with
high sperm count and motility
Highly viscous samples
respond poorly to the swim-
up technique
Contamination remains

20. DENSITY GRADIENT CENTRIFIGATION
Density gradient centrifugation (DGC)
DGC consists of centrifugation of semen overlaid on the density
gradient layers containing silane coated colloidal silica.
Centrifugation migrates the cells and accumulate them in different
gradient levels by density.
Normal sperm have a greater density than the abnormal sperm due to
their highly condensed DNA.
This is known as isopycnic centrifugation, the cells are accumulated at
the point where density is identical to the gradient media.
Discontinuous density gradient –a simple two step DDG is widely
applied.

21. DISCONTINUOUS 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
Anti sperm Antibodies
Highly viscous samples

22. DISCONTINUOUS DENSITY GRADIENT
Pipette 1ml of 80% density gradient medium into a
conical tube.
Slowly layer 1ml of 40% density gradient medium on
top of it.
Gently layer 1ml of semen sample on top of the 40%
layer.
Avoid adding too much of semen as it will results poor
separation.
Centrifuge at 2000rpm for 10 minutes.
Discard the top two layers.
Loose the pellet and transfer into a
sterile conical tube.

23. DISCONTINUOUS DENSITY GRADIENT
Resuspend the pellet with 3 ml of IVF media.
Centrifuge again at 1500rpm for 5 minutes.
Discard the supernatant and resuspend the pellet
with 0.5 -1ml of IVF media.
Assess count and motility and use for IUI.
Once the sample is ready, the sample
should be inseminate within 30 min.

24. DISCONTINUOUS DENSITY GRADIENT

25. DISCONTINUOUS DENSITY GRADIENT

26. DISCONTINUOUS DENSITYGRADIENT
ADVANTAGES:
Rapid separation technique
Require less time
Simple to perform
Abnormal sperms, immotile sperms and debris are largely
eliminated
Free from contamination and other seminal constituents.
Disadvantages:
Costly, technical skills

27. Retrograde Ejaculation Samples

28. Retrograde Ejaculation Samples
Alkalinization treatment given prior to sperm collection
No alkalinization treatment given prior to sperm
collection

29. Retrograde Ejaculation Samples
Alkalinization treatment
Drinking water with NaCl and
NaHCO
3 1-2 hours prior to
attempt
At the lab:
Produce an ejaculate by
masturbation into specimen
container
Urinate after orgasm in a
second container (~500ml)
Non alkalinization
treatment
At the lab:
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

30. RETROGRADE EJACULATION
SAMPLE
Centrifuge the whole content at 1500 rpm
for 5-8 minutes.
Discard the supernatant.
Once concentrated, either DGC/ Swim-up
can be performed.
Incubate it for 30 minutes and can be used
for insemination purposes.

31. Unconventional methods
Migration sedimentation
Glass wool filtration
Transmembrane migration

32. Newer techniquics
MACS-Magnetic activated cell sorting
Microfluids
Hyaluronan binding (PICSI, sperm slow)
Migration sedimentation device

33. MACS
The apoptotic and necroptotic sperms cell surface release
the phosphatidylserine (PS)
The caspase mediated apoptotic PS exposure is
irreversible
PS can be detected by annexin-V immunolabeling, which
has a high affinity to PS
Principle is selecting out the apoptotic sperm which are
attached to annexin-V
MACS is followed by DGC and swim-up yielded sperm
with low level of DNA fragmentation

34. MACS

35. MACS

36. MACS

37. MACS

38. MACS
Used in patients with high DNA fragmentation,
teratozoospermia, varicocele and couples with
unexplained infertility
Cochrane database systematic review reported that the
beneficial potential of MACS on clinical pregnancy, live
birth and miscarriage rates is uncertain and quality of
evidence is low.
The procedure is costly and has to be done in
combination with DGC.
No perinatal side effects were observed in this method

39. Microfluidics
Developed to imitate the sperm movements in the female
reproductive tract
Most of the devices create static micro channel pathways making
selections based on the ability of sperm to pass through labyrinth-like
paths depending on their motility
Taxis mediated
Chemotaxis-response to chemicals in the female tract or COC, e.g.
progesterone
Rheotaxis: Sperm swim against resistance towards the opposite
direction
Thermotaxis: Sperm express sensory receptors to detect temp
gradients e.g.: TRPV1 heat sensing receptor

40. Microfluidics

41. Microfluidics
Favorable in advanced maternal age and severe
oligozoospermia
Insufficient data regarding the superiority of microfluids
sperm selection
No significant results in ICSI cycles, unexplained couples,
sibling oocytes
More RCTs are required in this feild

42. Sperm selection by hyaluronic acid
binding
The mature sperm express HA binding sites which
attaches to HA coated surfaces from their head regions
HA binding sperms have higher capacity to bind to the
zona pellucida, higher fertilization ability, advanced
chromatin maturation, lower DNA fragmentation, lower
aneuploidy rate and superior morphology
P-ICSI
Spermslow

43. Sperm selection by hyaluronic acid
binding
P-ICSI(Physiological selection of sperm for ICSI)
Principle: solid state HA-coated areas on the surface of the classical
ICSI dish
The sperm with the high potential adhere to these surfaces whilst
passing through these areas
Sperms adhered to the coating are collected into the microinjection
needle and used for ICSI

44. Sperm selection by hyaluronic acid
binding
SpermSlow is a semi viscous medium containing hyaluronan and
allowing for slowing down the movement and selection of mature
spermatozoa with better DNA quality for ICSI
•No need for PVP during ICSI
•Significantly lower abnormal fertilization rate
•Significantly better embryo quality compared to standard ICSI
•Significantly higher implantation rate compared to standard ICSI
•Spermatozoa, selected with SpermSlow have significantly lower DNA
fragmentation rate
•Easy to implement in clinical practice

45. Sperm selection by hyaluronic acid
binding

46. Sperm selection by hyaluronic acid
binding
It is sperm selection method during ICSI only
Results compared with conventional ICSI related to high DNA
fragmentation, teratozoospermia, recurrent pregnancy loss-but
the results are conflicting
P-ICSI vs conventional ICSI-in RCTs no difference in results
Beneficial in old maternal age groups
Sibling oocytes with previous failure –improved fertilization and
embryo utilization rates in p-ICSI group
Further studies needed to establish which patients may benefit
from this technique

47. Migration sedimentation device

48. VIRAL INFECTED SAMPLES
Treatment methods that increase the chance of
pregnancy
Reduce the risk of horizontal (person to person) viral
transmission
Reduce the risk of vertical (mother to baby) viral
transmission
HIV, HBV, HCV
Improved anti-viral medications has increased life
expectancy and quality of life for patients with BBVS

49. HIV INFECTED SAMPLES
Viral RNA and proviral DNA can be found in seminal plasma and in
non-sperm cells.
HIV receptors (CD4, CCR5,CXCR4) are expressed by non-sperm cells
DGC followed by swim-up is the best way of preventing infection in
uninfected female partners
This procedure aims at separating virus infected non-sperm cells and
seminal plasma from HIV free, motile sperms in the swim-up
Prepared samples should be tested by RT-PCR before use and only HIV-
free samples should be used for ART.
So far the results are encouraging still insufficient evidence of the
elimination on risk of HIV infection through sperm preparation

50. Hum Reprod Open, Volume 2021, Issue 4, 2021, hoab037, https://doi.org/10.1093/hropen/hoab037
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Summary of management of medically assisted reproduction in
patients testing positive for HIV

51. HCV/HBV INFECTED SAMPLES
HCV
DGC followed by swim-up is the best way of preventing
infection in uninfected female partners
PCR testing may not be necessary
HBV
Currently no sperm preparation techniques can select
HBV DNA-free spermatozoa
Theoretical risk of vertical transmission remains a
possibility

52. Hum Reprod Open, Volume 2021, Issue 4, 2021, hoab037, https://doi.org/10.1093/hropen/hoab037
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Summary of management of medically assisted reproduction in
patients testing positive for hepatitis C virus.

53. Hum Reprod Open, Volume 2021, Issue 4, 2021, hoab037, https://doi.org/10.1093/hropen/hoab037
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Summary of management of medically assisted reproduction in patients
testing positive for hepatitis B virus

54. Hum Reprod Open, Volume 2021, Issue 4, 2021, hoab037, https://doi.org/10.1093/hropen/hoab037
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VIRAL INFECTED SAMPLES

55. Testicular/epidydimal samples
Azoospermia –testicular or epididymal aspirate fluid can
be obtained from the male genital tract by
MESA-Microsurgical Epididymal Sperm Aspiration
PESA-Percutaneous Epididymal Sperm Aspiration
TESA-Testicular Sperm Aspiration
TESE-Testicular Sperm Etraction
mTESE-micro-TESE

56. Testicular/epidydimal samples

57. Testicular/epidydimal samples
Testicular specimens are invariable contaminated with non germ
cells and large number of red blood cells
So additional steps are needed to isolate a clean preparation of
spermatozoa
To free the seminiferous tubule bound testicular sperm
enzymaticor mechanicalmethods are needed
ICSI is preferred as sperms are low in number and poor motility

58. Testicular/epidydimal samples
Enzymatic method
It involves incubating the testicular tissue with collagenase for 1.5-2
hours at 37 °c and vortexing the suspension every 30 minutes
Mechanical method
It involves the maceration of the testicular tissue in the culture medium
using either glass cover slips or fine needles that are bent parallel to
the base of the culture dish until a fine slurry of dissociated tissue is
produced.
It is recommended DGC method to obtain clean preparations.
Simple washing in case of low sperms

59. Testicular/epidydimal samples

60. Assisted ejaculation samples
Who cannot ejaculate / may be collected by direct
vibratory stimulation of the penis or rectal electrical
stimulation.
Spinal cord injury
They frequently have high sperm concentrations,
decreased sperm motility, red or white blood
contamination
DGC is most effective method
Regardless of the method of preparation they often have
high percentage of immotile sperms

61. SEMEN SAMPLES PRE AND POST
WASH
PREWASH SAMPLE POSTWASH SAMPLE

62. Key points
Swim-up and DGC are the best standardize methods for
the sperm preparation
Retrograde samples –concentrate the sample followed
by DGC-ICSI preferred
NEWER TECHNIQUES:
MACS-Mixed results with MACS, may be useful in
particular category of patients
high DFI, apoptotic sperms, teratozoospermia,
unexplained infertility

63. Key points
MICROFLUIDCS-high DFI, high maternal age,
teratozoospermia samples
HB-High DFI, Teratozoospermia, Recurrent pregnancy
loss
New gadgets-additional cost-IVF results ?

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KEY POINTS

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KEY POINTS

66. KEY POINTS
Testicular sperms/epididymal sperms
TESA/PESA/MESA/TESE/m-TESE
Enzymatic/ mechanical-followed by DGC
For immotile sperms in testicular tissue-treatment with
pentoxifylline/theophylline/HOST
In assisted ejaculation –where long abstinence-DGC
preferred-low motility

67. PRECAUTIONS
Damage to the sperm from dilution, temperature change,
centrifugation, and exposure to potentially toxic material
must be minimized
Dilution should be perform slowly
Temperature changes should be gradual
Preparation should be perform at 37°c/RT
Duration of centrifugation rather than the centrifugation
speed leads to higher ROS Production.
Optimum sperm incubation time before sperm
preparation is 20 min.

68. Take home message
“ An ideal sperm preparation technique should
recover a highly functional sperm population that
preserves DNA and does not induce dysfunction
through the production of ROS by sperm and
leukocytes”