ICSI as it is presently performed is far from an ideal solution because the selection of sperm is based on the judgement of an embryologist, who is looking for the most normal appearing sperm available.

1. SPERM SELECTION METHODS
Dr. Rahul

2. DAY 0 1 2 3 4 5
early development later development
• Oocyte activation factor
• Organelle Factors
• Membrane – cytosolic
factors
• Cytoplasmic Factors
• Nuclear Factors
• DNA-fragmentation - integrity
Impact of sperm over embryo development
EMBRYONIC DEVELOPMENT IMPLANTATION RATES PREGNANCY RATES MISCARRIAGE

3. Why sperm cells are interesting…???
• Almost always in
Excess: not a
limiting factor
• Genetic
uniqueness in
each sperm
provides a wide
variety of
phenotype
(Wang J, Fan HC, Behr B, Quake SR.
The Cell. 2012 )
• During in vivo
we do not know
how one sperm
is selected and
on what basis

4. SUCCESS

5. In vitro sperm selection
• Semen examination
• Morphology assessment
• Sperm preparation methods
• Sperm Function tests
• Sperm selection in viscous medium
• Advanced methods of sperm selection

6. Semen Examination
• Hallmark for fertility evaluation in
male
• Provides more detailed
information on routine basis
• Abnormalities in sperm
production or function, alone or
in combination with other factors,
account for 30–50% of all cases
of infertility outcomes(W.H.O, 2010).

7. SEMEN EXAMINATION
Macroscopic parameters
• Volume
• Appearance
• Color
• Liquefaction
• Viscosity
• pH
Microscopic parameters
• Sperm concentration
• Sperm motility
• Morphology
• Leucocyte concentration
• agglutination

8. Cut off reference values for sperm parameters in
consecutive WHO manuals

9. Sperm morphology
• In the 80’s, pioneer studies on sperm
morphology (Tygerberg’s, SA & Kruger,
USA) correlated the architecture of
spermatozoa with IVF outcomes &
suggesting importance.
• Predictive value of sperm morphology
for fertilization and pregnancy
outcomes in IVF treatments. Parinaud
et al., 1993; Ombelet et al., 1997;
Eilish T. et al., 1998
• Several ways to analyze sperm
morphology
• All morphological measurements to be
made using fixed stained smears
(Papaniculao OR Diff-Quick OR Shorr-
stain)

10. Krugers et. Al., 1988
HEAD
SMOOTH, OVAL CONFIGURATION; LENGTH 4-6um, DIAMETER-2.5-3.5 um,
ACROSOME MUST CONSTITUTE 40-70% OF SPERM HEAD
NECK/
MIDPIECE
SLENDER, AXIALLY ATTATCHED, 1.5 TIMES OF HEAD LENGHT, SLIGHTLY
THICKER THAN TAIL
TAIL SINGLE, UNBROKEN, STRAIGHT, WITHOUT KINKS OR COILS
KRUGERS STRICT CRITERIA
NORMAL SPERM
ABNORMAL SPERMS
AMORPHOUS
HEAD
NECK
DEFECT ABNORMAL ACROSOME FORMS

12. Semen preparation
• Semen composed of
❖ Mixture of secretions, other cells and many anti capacitating factors
❖ Risk of oxidative & osmotic damage in vitro
❖ Needed to separate best motile and morphologically normal sperm
Classical sperm preparation methods are based on motility & counts.
·Migration ·Swim Up ·Gradient centrifugation

13. SPERM FUNCTION TESTS
HYPO-OSMOTIC SWELLING TEST
• Based on the principle that
viable spermatozoa have intact
membranes.
• Characteristics: Swelling of the
cytoplasmic space and curling
of the sperm tail.
• Normal test results: >58%
curling tail is considered normal.
• Test may be used to aid
selection of viable spermatozoa
for use in ICSI, especially in
those cases of severe
asthenospermia.
DNA FRAGMENTATION INDEX
• Susceptibility of DNA damage in vitro is High
• Important causes of reduced fertility
potential
• Useful marker for sperm Function (Aitken et
al., 2010)
• High sperm DNA fragmentation values have
negative impact on ART outcomes (lopez G
et al., 2013 Simon Let. Al., 2010)
• Various tests available to analyze sperm DFI
on the basis of Strand Breaks, Susceptibility
to Denaturation, Chromatin Condensation
• Unclear thresholds, less sensitive,
labor intensive & variable protocols

14. Sperm Motion
• Not easy to select sperm
according to morphological
characteristics while they are
in motion under culture media.
• Can be accomplished by
observing its shape, light
refraction and motion pattern
in viscous medium
• The viscous medium
decelerate spermatozoa
allows evaluation in its
tridimensional motion pattern,
permitting morphological
assessment.
PVP
MOPS

15. Surgically retrieved sperms
Gentle Dissection of ST
Resuspension> Rinse> HOS/PVP> ICSI © ANDROFERT 2010 , Sandro C Esteves

16. Advanced sperm
selection methods

17. IMSI
• Bartoov et al introduced ICSI using high-power
differential interference contrast optics, allowing
visualization of sperm at ultra high magnification & in
real time.
• Fine morphological integrity of human sperm nuclei is
positively associated with fertilization and pregnancy
rates.
• The strict descriptive criteria for normally shaped
nuclei (Bartoov et. Al., 2001, 2003)
ROUTINE ICSI 200x-400x IMSI 7000x

18. Criteria for morphologically normal nucleus
-Smooth, Symmetric & Oval shape
-Nucleus- homogeneous, no extrusions, no invaginations,
- maximum of one vacuole less than 4% of the nuclear area.
Bartoov et al., 2001
(MSOME)Motile Sperm Organellar Morphology Examination
Normal sperms Abnormal sperms

19. Study Significant Findings (IMSI vs. ICSI)
Souza Setti et al, 2010 Significantly higher implantation and pregnancy, significantly
lower miscarriage rate in IMSI group
Figueira et al, 2010 Significantly lower aneuploidy rate in IMSI group
Wilding et al, 2010 64.8% of sperm selected in ICSI had significant DNA
fragmentation. Embryo quality, pregnancy and implantation
rate higher in IMSI group
Monquat et al, 2010 Swim up sperm have less nuclear vacuolation than DGS
Vanderzwalmen et al,
2008
Presence of nuclear vacuoles reduces PR, higher pregnancy
rate in IMSI group. Proposed sperm grading scheme
Antinori et al 2008 +ve correlation for pregnancy and miscarriage in OAT group
Bartoov et al, 2002 Significantly higher pregnancy rate in IMSI group
Bartoov et al, 2002 Describe MSOME. Sperm with vacuole occupying >4% of
nuclear area abnormal and not used for injection
Bartoov, 2001 First reported IMSI. Magnification x6000.

20. Meta-analysis

21. PICSI- Physiological ICSI
• In the process of fertilization, the sperm
bind to the hyaluronic acid (HA) present in
the cumulus oophorus by HspA2 protein,
with hyaluronidase activity that induces
acrosome reaction.
• The PICSI device, a dish similar to ICSI dish,
contains microdots of hyaluronan hydrogel
which needs to be hydrated by media
before ICSI.
• Selectively binding of sperm to the culture
dish shows:
More viability,
Non DNA-fragmented, and
with lower rates of aneuploidies.

22. Selection of HA binding spermatozoa
Incubation, RT, 10
min
Add sperm to the hyaluronan
microdot
Gentle aspiration of a bound
spermatozoa
After 15 min, the proportion of hyaluronan-bound spermatozoa, that exhibited vigorous tail
beating, were assessed versus the unbound motile spermatozoa

23. © Fert & Steril, 2011

24. MACS (Magnetic Activated Cell sorting)

26. Advanced sperm selection techniques are
hypothesized to improve ART outcome through
the selection of sperm with a variety of
“beneficial characteristics.”
Although individual small studies have suggested
that these techniques have clinical benefit, there
remains less comprehensive review of
randomized controlled trials (RCTs).
The current review needs more studies to be
included, so the results can better guide clinical
practice and further application.

27. The gold standard of sperm function
remains the ability to fertilize an oocyte
in vitro.
Abnormal shaped sperm have an
increased chances of poor outcomes.
Proper semen examination, sperm
preparation and wise sperm selection
technique recommended in ART.
The onus for sperm selection shifted
from counts, motility to other
parameters.

28. The Key to everything is Patience.
You Get the Chicken by Hatching the egg, not by smashing it.
- Arnold H Glasow
Thanking You ALL…