This document discusses sperm sorting techniques for men with high sperm DNA fragmentation index. It begins by defining sperm DNA damage and fragmentation, then discusses causes and indications for testing. It describes different tests to measure sperm DNA fragmentation and diagnostic cut-off points. The objectives and limitations of sperm sorting are outlined. Various sperm sorting techniques are explained, including swim-up, density gradient, magnetic activated cell sorting (MACS), motile sperm organelle morphology examination (MSOME), and surface charge-based sorting using hyaluronan binding. Advanced techniques like MSOME and MACS aim to select sperm with intact DNA and normal morphology to improve fertility outcomes.

1. Sperm Sorting techniques in
high Sperm DNA Fragmentation Index
Jindal IVF & Sant Memorial Nursing Home
Chandigarh India

2. Introduction
Sperm DNA damage can be defined as any
chemical change in the normal structure of the
DNA.
Sperm DNA fragmentation (sDF) is one of the
most common disturbances affecting the
genetic material in the form of single or double
strand breaks.

3. DNA damage Mechanism
Abnormal chromatin packaging
Reactive oxygen species
Abortive apoptosis

4. Indications of Sperm DNA
Fragmentation Testing
• Unexplained or persistent infertility
• Failure to conceive after 5-6 intra uterine insemination (IUI) cycles
despite good count and motility
• Low fertilization rates or poor embryo quality in IVF cycles
• Recurrent miscarriage
• Prolonged stay in an environment that exposes to reproductive
toxins
• Abnormal semen analysis
• Advancing male age (>45 years)
• Smokers

5. Types Sperm DNA Fragmentation Testing
• Sperm Chromatin Dispersion Test
• Acridine orange
Sperm
Chromatin
Dispersion Test
• Sperm Chromatin Structure Assay
• TUNEL Assay
(Terminal deoxynucleotidyl transferase
mediated dUTP nick end-labeling)
Flow Cytometry
• Comet Assay
(Single-cell gel
Electrophoresis)
Electrophoresis

6. Testing Cut off Ranges
• < 30%
Sperm
Chromatin
Dispersion Test
• <20%
Flow Cytometry
(TUNEL)
• < 80%
Electrophoresis
(Comet Assay)

7. Prognostic value of sperm DNA damage in
predicting clinical pregnancy
L. Simon, L. Liu, K. Murphy, S. Ge, J. Hotaling, K.I. Aston, B.Emery,
and D.T. Carrell: Human Reproduction, Vol.29, No.5 pp. 904–917, 2014

8. Best method for
Sperm DNA Fragmentation

9. Diagnostic cut-off point at JISNH
DFI Fertility Potential
< 15 % Excellent
15-30 % Good
> 30 % Poor

10. Objectives of Sperm sorting
To maximize the chances of fertilization by obtaining
SPERMATOZOA with the highest potential for fertilization
from semen sample by
1) Removal of prostaglandins.
2) Removal of pathogens.
3) Removal of antibodies.
4) Removal of debris & dead spermatozoa.
It cannot increase or improve
the basic sperm quality or number

11. The final outcome is to select viable, motile and
morphologically intact sperm by significantly reducing the
percentage of spermatozoa with nuclear abnormalities and
DNA damage.
Final Outcome

12. Sperm Sorting
 For IUI
 For IVF
 For ICSI

13. Selection of Technique
Ideal technique
 Quick, easy and cost-effective.
 Isolate as much motile sperm as
possible.
 No sperm damage or physiological
alterations.
 Eliminate dead spermatozoa and
non sperm elements, toxic or
bioactive substances like
decapacitation factors or ROS.
 Allow processing of larger volumes
of ejaculates
Proper technique
 Since none of the methods
available meets all these
requirements, a variety of
sperm separation techniques is
mandatory in clinical practice
to obtain an optimal yield of
functionally competent
spermatozoa for insemination
purposes. Depending on the
ejaculate quality, these
methods have different
efficiency and areas of use

14. Functional & Molecular Assay
 Capacitation tests
 Zona-free hamster penetration assay
 Membrane integrity tests
 Antisperm antibodies
 Vital staining
 Biochemical analysis
 Peroxidase staining
 Sperm ubuquitin tag
immunoassay (SUTI)
 Semen culture
 Hypo-osmotic swelling test
 Sperm penetration assay
Hemizona assay
 Creatinkinase
 Reactive Oxygen Species (ROS)
 CASA

15. Types of Techniques
Conventional Advanced

16. Progress so far in widely adopted in
clinical practice
B. Direct- Swim up C. Density Gradient
A. Simple Washing

17. Swim-up
• Based on motility.
•Used for IUI
•Only for good semen
sample
•Sperm recovery is less

18. Density Gradient
• Based on adherence
due to density.
• Used for IUI/IVF/ICSI
• Used for samples with
pus cells etc
• High recovery of
sperm

19. DNA Fragmentation (%)
Raw
Sample
18.3±10.
8
Direct
Swim-up
6.6±5.7
Density
Grad.
12.2±9.9
Density
Grad.
Swim-up
3.7±4.9
Pellet
Swim-up
4.2±3.8
Aldo Volpes, Francesca Sammartano, Simona Rizzari,
Salvatore Gullo, Angelo Marino, Adolfo Allegra. Journal of
Assisted Reproduction and Genetics;
Gamete Biology DOI 10.1007/s10815-016-0696-2, First
online: 16 March 2016

20. Traditional sperm parameters & ICSI
Success of ICSI independent of traditional sperm parameters
Hum Reprod, 1995

21. New sperm parameters & ICSI
Fertil Steril, 2008
New sperm parameters are associated with ICSI outcome

22. Advanced Techniques
MSOME (IMSI)
MACS
SURFACE CHARGE BASED
SPERM HEAD BIREFRINGENCE
GLASS WOOL FILTRATION
PICSI
MICROFLUIDICS

23. MSOME
(Motile Sperm Organelle Morph. Exam)
 Examination performed in real time on living
Sperm.
 Inverted light microscope
 Equipped with high-power Nomarski optics
instead of Hoffman Modulation Contrast
 Enhanced by digital imaging to achieve a
magnification up to 6300.
 More accurate examination of spermatozoa

24. MSOME
>6600X Nomarski
Bartoov et al.,
ICSI
2-400 X Hoffman
Palermo et al., 1992
Equipment for MSOME

25. Motile Sperm Organelle Morphology
Examination -MSOME

26. Criteria
Motile sperm organellar morphology examination (MSOME)
Bartoov et al., 2002
Based on ultrastructural studies of
acrosome, postacrosomal lamina, neck,
mitocondria, tail and nucleus Glezerman
and Bartoov, 1993; Bartoov et al., 1994
Head shape normal = Oval, smooth and
symmetrical, size 4.75 ± 0.28 μm (length)
to
3.28 ± 0.20 μm (width)
Chromatin content normal= no vacuoles or
the vacuoles occupy < 4% of the nuclear
surface (0.78 ± 0.18 μm)
No vacuoles or
less than 4%
of the nuclear
surface

27. VACUOLES
 Association between vacuoles and sperm
DNA fragmentation (Garolla et al., 2008;
Franco et al., 2008; Berkovitz et al., 2005;
Oliveira et al., 2010)
 Association between vacuoles and
aneuploidy (Garolla et al., 2008)
 Lower risk of sex chromosome
abnormalities in IMSI vs. IVF (OR 0.57,
CI 0.37-0.90) Figueira et al., 2011

28. Vacuoles
Fertilization & embryo development
 Vacuoles associated with lower fertilization rates (Cassuto et
al., 2009; Franco et al., 2008)
 Correlation between presence and size of vacuoles and
cleavage stage embryo development (Berkovitz et al., 2005;
2006a,b)
 No correlation between presence and size of vacuoles and
cleavage stage embryo development (Hazout et al., 2006;
Antinori et al., 2009; Mauri et al., 2010; Balaban et al.,
2011)
 Correlation between presence and size of vacuoles and
blastocyst development Vanderzwalmen et al., 2008

29. IMSI vs. ICSI
Implantation and pregnancy
 Implantation and pregnancy results higher in IMSI
vs ICSI group Bartoov et al., 2003; Berkovitz et al., 2005; Hazout
et al 2006; Berkovitz et al., 2006a, 2006b;
 Abortion rates lower in IMSI vs. ICSI group Bartoov et
al., 2003; Berkovitz et al., 2005; Hazout et al 2006; Berkovitz et al.,
2006a, 2006b

30. Magnetic Activated Cell Sorting

31. What is MACS?
Magnetic activated cell sorting of
human spermatozoa
 An advanced sperm preparation
technique working on the principle
to separate sperm cells with
apoptotic features .

32. Apoptosis
 Apoptosis is the programmed cell death that occurs because of the DNA
fragmentation which is seen in the sperm of infertile men.
 Sperm cells with apoptotic features can remain normally shaped
 They may still be able to fertilize an oocyte.

33. MACS: Technique
•Semen sample is mixed with
supraparamagnetic beads
conjugated to highly specific
antibodies to annexin-V are
incubated at room temperature
for 15 minutes .
•The mixture is loaded on top of
separation column which is
placed in the magnetic field

34. MACS
Pros
 Rapid, convenient, non invasive
 Acts at molecular level
 Only technique that separates
apoptotic sperm
 Provides optimal purity and
reliable and consistent results
 Optimise cryopreservation thawing
outcomes
Cons
 MACS need to be used in
conjunction with other technique
such as DGC to remove seminal
plasma

35. Sperm selection based on surface
charge
A. Electrophoretic method
B. Zeta potential

36. Principle
 Mature spermatozoa carry an electronegative surface
charge, which is attributed to sialic acid residues
including CD52 found on sperm plasma membrane.
 CD52 is acquired during epididymal maturation. Its
presence indicates normal sperm morphology and
capacitation; therefore, it can be considered as an
indication of sperm maturity and quality.
Electrophoresis technique
 External electrical current is applied and mature
negatively charged spermatozoa moves towards
positive electrode

38. Hyaluronan Binding Theory
 Mature sperm have completed the
plasma membrane remodeling and
have receptors for and canbind
to hyaluronic acid (HA)
 Immature sperm have not developed
receptors for hyaluronic acid and will
not bind to HA

39. Hyaluronan Binding Theory
A sperms ability to bind to HA correlates to:
 Kruger Strict Morphology
 Cellular maturity,
 Less rates of chromosomal aneuploidy,
 Less rates of DNA fragmentation,
 Increased chromatin integrity
 Normal head morphology = better fertilizing potential

40. Hyaluronan (Hyaluronic acid, HA):
 Is an anionic, non-sulfated glycosaminoglycan
 Is the major component of the Cumulus Oophorus complex surrounding the human oocyte.
 Provides viscoelastic properties for the cumulus structures
 HA assists the binding of the cumulus cells together and importantly acts to activate the sperm
whilst in the cumulus.
What is Hyaluronan ?

41. Low HBA score ≤65% :
• Decreased quality of sperm sample = evaluate further treatment (IUI/IVF/ICSI)
• Decreased likelihood of selecting best sperm for ICSI using morphology
evaluation only
HB Assay – Hyaluronan Binding Assay Diagnostic tool
- Sperm sample evaluation in minutes
• Is an important diagnostic tool used in the analysis of semen.
• In a matter of minutes it provides an answer to the proportion of mature sperm in the
sample (HBA score).
• ONLY mature sperm with developed HA receptors bind

42. HB Assay – Hyaluronan Binding Assay Diagnostic tool
Designed with two duplicate
chambers coated with HA

43. Mature spermatozoa show a reduction of more than five fold in
aneupliody rate than immature ones.

44. 44
Number of samples Analysis
Sperm DNA integrity
in semen and in their
respective HA-bound
sperm fractions was
studied in 50 men
Proportions of sperm with green
AOF (high DNA integrity) and red
AOF (DNA breaks) were
evaluated by fluorescence
microscopy
Conclusion
Sperm selection of HA and of zona pellucida are similar in
selecting
sperm with high DNA integrity.

45. Number of samples Analysis Conclusion
19 sperm samples;
husbands from infertile
couples
HBA ”unbound” fraction
vs TUNEL
Statistical correlation between the
percentages of HA-unbound sperm and
TUNEL positive sperm.

46. Number of samples Analysis Conclusion
192 patients undergoing ICSI Retrospective study comparing ICSI outcome
with HBA score
The higher the HBA score the higher the better
the fertilization, pregnancy and cleavage rates.

47. Number of samples Analysis
804 ICSI patients Patients were grouped according to HBA score
(above or below 65%), and in each group
subgroups had sperm selected for ICSI by PVP
(Control) or Hyaluronic acid
(HYAl group =PICSI dish)
Conclusion
Selecting spermatozoa for ICSI using Hyaluronic acid lead to a
significantly lower pregnancy loss rate in patients with low HBA score
(≤65%)

48. PICSI dish
• ICSI dish with hyaluronan coated dots for sperm selection
• Bound sperm = Mature sperm with high DNA integrity
• Benefit with PICSI increase as HBA score decrease (low binder samples)
Important:
Could reduce number of unexplained failures by preventing injection of “good-
looking” (but immature) sperm

49. PICSI
Pros
 Physiological/Natural process of
selecting sperm
Cons
 Sperm are selected individually
i.e. more demanding and longer
process .
 Used media can affect sperm
 Only for motile sperm

50. Sperm Slow
• Semi viscous medium containing Hyaluronic acid
• Natural alternative to PVP = ONLY slows sperm with HA
receptors (e.g. Mature sperm with high DNA integrity)
• PVP may induces nuclear damage and chromosomal
aberration.
• PVP injected into the egg along with the sperm cannot
diffuse out or be broken down intracellular & remain in the
developing embryo for a prolonged period, where it is likely
to impede embryo development and pregnancy.

51. Sperm Slow
SpermSlow
Clean medium
Sperm
suspension
Spermatozoa bound to HA in
the junction zone of the
droplets can be selected and
easily detached by injection
Holding
medium
Several studies looking into the benefits of using Hyaluronan
based sperm selection clearly finds several advantages
compared to regular sperm selection (PVP):
• Significantly higher Embryo Developmental Rate
• Better Embryo Quality
• Lower rates of DNA damage in HA- selected sperm
• Lower rates of early miscarriage
Parmigiani et al., (2010) Physioloic ICSI’’: Hyaluronic acid (HA) favors
selection of spermatozoa without DNA fragmentation and with
normal nucleus, resulting in improvement of embryo quality

52. Microfluidics
“MICROFLUIDIC” refers to technology utilizing characteristics of
fluid movement in a micro or nano environment.
Microfluidics for sperm sorting: Active selection
Active selection means a set of strategies using the active
swimming of the sperm cells that takes inspiration from the
natural selection that occurs in the female reproductive tract
based on mainly 3 phenomena:
• Chemotaxis
• Thermotaxis
• Rheotaxis.

53. PRINCIPLE
 At the macro-level, fluid flow results in chaotic particle movement within
the fluid stream, leading to turbulence.
 Microfluidic devices impose laminar flow upon fluids, allowing parallel
movement of multiple streams of media through the same microchannel with
no mixing, except by diffusion across the fluid–fluid interface.
 Gravity driven laminar flow of sperm suspension in microfluidic channels
allowing only motile spermatozoa to swim into parallel stream

54. Microfluidics

55. Microfluidics

56. ADVANTAGES
 No sample preparation
 No expensive equipment
 No centrifugation
 No extensive training
 Low ROS & DNA fragmentation
 Sterile, Single – Use chips
 Sperm sorting based on motility within a micro –channels or a micro – porous
filter.

57. Microfluidic sorting selects sperm for clinical use with reduced DNA
damage compared to density gradient centrifugation with swim-up in
split semen samples
Human Reproduction, Vol.33, No.8 pp. 1388–1393, 2018