Sperm Preparation in High DFI | Jindal IVF

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Sperm Sorting techniques in high Sperm DNA
Fragmentation Index

• Jindal IVF Sant Memorial Nursing Home
• Chandigarh India

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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.

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DNA damage Mechanism
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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 (gt45 years)
• Smokers

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Types Sperm DNA Fragmentation Testing
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Testing Cut off Ranges
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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. 904917, 2014
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Best method for Sperm DNA Fragmentation
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Diagnostic cut-off point at JISNH
DFI Fertility Potential
lt 15 Excellent
15-30 Good
gt 30 Poor
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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

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Final Outcome
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.
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Sperm Sorting

• For IUI
• For IVF
• For ICSI

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Selection of Technique

• Ideal technique

• Proper 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

• 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

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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

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

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

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Density Gradient

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

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DNA Fragmentation ()
Aldo Volpes, Francesca Sammartano, Simona
Rizzari, Salvatore Gullo, Angelo Marino, Adolfo
Allegra. Journal of Assisted Reproduction and
Genetics Gamete Biology DOI 10.1007/s10815-016-0
696-2, First online 16 March 2016
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Traditional sperm parameters ICSI
Success of ICSI independent of traditional sperm
parameters
Hum Reprod, 1995
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New sperm parameters ICSI
New sperm parameters are associated with ICSI
outcome
Fertil Steril, 2008
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Advanced Techniques
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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

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Equipment for MSOME
ICSI 2-400 X Hoffman Palermo et al., 1992
MSOME gt6600X Nomarski Bartoov et al.,
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Motile Sperm Organelle Morphology Examination
-MSOME
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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

No vacuoles or less than 4 of the
nuclear surface
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 lt 4 of the nuclear surface
(0.78 0.18 µm)
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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

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VacuolesFertilization 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

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IMSI vs. ICSIImplantation 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

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Magnetic Activated Cell Sorting
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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 .

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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.

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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

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MACS

• Pros

• Cons

• 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

• MACS need to be used in conjunction with other
  technique such as DGC to remove seminal plasma

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Sperm selection based on surface charge

1. Electrophoretic method
2. Zeta potential

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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

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Hyaluronan Binding Theory

• Mature sperm have completed the plasma membrane
  remodeling and have receptors for and can bind to
  hyaluronic acid (HA)
• Immature sperm have not developed receptors for
  hyaluronic acid and will not bind to HA

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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

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What is Hyaluronan ?

• 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.

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•  
• 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

• 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

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HB Assay Hyaluronan Binding Assay Diagnostic
tool
Designed with two duplicate chambers coated with
HA
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Mature spermatozoa show a reduction of more than
five fold in aneupliody rate than immature ones.
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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 Conclusion
Sperm selection of HA and of zona pellucida are similar in selecting sperm with high DNA integrity. Sperm selection of HA and of zona pellucida are similar in selecting sperm with high DNA integrity.
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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.
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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.
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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 Conclusion
Selecting spermatozoa for ICSI using Hyaluronic acid lead to a significantly lower pregnancy loss rate in patients with low HBA score (65) Selecting spermatozoa for ICSI using Hyaluronic acid lead to a significantly lower pregnancy loss rate in patients with low HBA score (65)
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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
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PICSI

• Pros

• Cons

• Physiological/Natural process of selecting sperm

• Sperm are selected individually i.e. more
  demanding and longer process .
• Used media can affect sperm
• Only for motile sperm

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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.

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Sperm Slow

• Several studies looking into the benefits of
  using Hyaluronan
• based sperm selection clearly finds several
  advantages
• compared to regular sperm selection (PVP)
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• Significantly higher Embryo Developmental Rate
• Better Embryo Quality
• Lower rates of DNA damage in HA- selected sperm
• Lower rates of early miscarriage

Holding medium
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
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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.

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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
  fluidfluid interface.
• Gravity driven laminar flow of sperm suspension
  in microfluidic channels allowing only motile
  spermatozoa to swim into parallel stream

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Microfluidics
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Microfluidics
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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.

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Microfluidic sorting selects sperm for clinical
use with reduced DNA damage compared to density
gradient centrifugation with swim-up in split
semen samplesHuman Reproduction, Vol.33, No.8
pp. 13881393, 2018