insight to ivf a laboratory perspective - fertility society · assist in preparing gametes for...
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Insight to IVF ndash A Laboratory Perspective
Laboratory Environment
bull Need homeostatic non-toxic nurturing environment
bull Warm laboratory to minimise temperature fluctuations
bull Assure nothing in lab is toxic
bull Keep lab free of VOCs ndash No cardboard in lab
ndash Air new equipment outside of lab
ndash Use VOC free paints
ndash Check VOCs using VOC measuring device
bull Take care with cleaning reagents within the laboratory
bull Embryologists contributing to environment ndash No perfume lotions make up
IVF Laboratory
bull Optimal culture conditions for optimal pregnancy results
bull Commitment to Single Embryo Transfer
bull Embryo culture
bull Cryopreservation of sperm eggs embryos ovarian tissue
bull Manage patient expectations through flexibility and choice
Function of the Embryology Laboratory
bull Culture media preparation
bull Egg collections (oocyte retrieval egg harvesting EPU VPU)
bull Sperm Preparation (and sometimes extraction)
bull Insemination
bull Embryo Culture
bull Embryo biopsy (as required)
bull Embryo transfer
bull Gameteembryo freezing and thawingvitrification and
warming
ndash With respect to our patients
ndash With the highest attention to detail
ndash With extensive witnessing procedures
Culture conditions and quality control bull Most common Complex Sequential Culture media used
ndash Complex means it provides all nutrients including amino acids and
protein for optimal embryo growth
ndash Sequential as it mimics the environment in the fallopian tubes then
uterus thus the culture media embryos are exposed to is changed at
appropriate times of development adapting to embryorsquos
requirements
bull Importance of temperature and pH
ndash Controlled environment essential for optimal
embryo growth therefore we use
bull IVF Chambers
bull Mincs
bull Strict QCQA Program
What influence does specific culture media and
environment have on our embryos
What is our end point in IVF
What is in culture media
bull Water
bull Salts
bull Energy substrates glucose pyruvate lactate
bull Buffer NaHCO3MOPs
bull Non-essential Amino Acids
bull Essential Amino Acids
bull Glutamine
bull Chelators ndash mainly EDTA
bull Macromolecules AlbuminHyaluronan
bull Antibiotics
bull Phenol Red
What is the purpose of culture media
Assist in preparing gametes for fertilisation and
grow embryos to then be placed into uterus
Differences between oviduct and uterus in mammalian embryos
Component Oviduct Uterus
Glucose concentration 05 mM 315 mM
Pyruvate concentration 032 mM 010 mM
Lactate concentration 105 mM 52 mM
Oxygen concentration 8 15
Carbon dioxide conc 12 10
pH 75 71
Glycine concentration 277 1933
Alanine concentration 05 124
Serine concentration 03 2080
Differences in the physiology of the mammalian embryo for
development from the zygote to the blastocyst stage
Precompaction stage Postcompaction stage
Low biosynthetic activity High biosynthetic activity
Low QO2 (Metabolic Quotient) High QO2 (Metabolic Quotient)
Pyruvate-based metabolism Glucose-based metabolism
Maternal genome Embryonic genome
Single cell Transporting epithelium
Low ability to maintain cellular Complex systems for maintenance
homeostasis of cellular homeostasis
Totipotent Differentiation into inner cell mass
and trophectoderm
Oviduct Uterus
uarr pyruvate darrpyruvate
uarrlactate darr lactate
darrglucose uarr glucose
uarrnon-ess AA uarr non-ess AA
uarr essential AA
Does culture media matter if your patients are
getting pregnant
bull Evidence that there are differences in DNA methylation patterns in
placental and umbilical blood samples from IVF children
compared to naturally conceived
bull There is an association with ART and specific imprinting disorders
bull SubfertilityInfertility hormone stimulation in vitro culture uterine
environment appear to influence the proper establishment and
maintenance of genomic imprints in the developing epigenome
Epigenetics amp Imprinting
bull Epigenetics Chromatin modifications that regulate gene activity
that are not due to DNA sequence changes (Saitou et al 2012)
bull Imprinted genes are genes whose expression is determined by
the parent that contributed them
bull Imprinted genes violate the usual rule of inheritance that both
alleles in a heterozygote are equally expressed
bull Genomic imprinting is a specialized epigenetic mechanism that
employs repressive modifications to silence one parental allele
while activating modifications on the other parental allele enable
expression Hirasawa amp Feil 2010
bull DNA methylation and histone modifications are two epigenetic
mechanisms that alter the functional state of chromatin activating
or repressing gene expression
In Vitro Embryo culture ndash Culture Media
bull Does culture media effect the health of the embryo
bull Suboptimal media may compromising imprint maintenance
ndash Mouse embryos cultured in deficient media displayed
biallelicH19 expression in postimplantation extraembryonic tissues
(Sasaki et al 1995)
ndash Whittens but not potassium simplex optimized medium plus amino acids
(KSOMaa) cultured embryos exhibited biallelic H19 expression
(Doherty et al 2000)
bull Extremely important for culture media to provide all the nutritional
requirements when required
bull Metabolites and cofactors are important regulators of the
epigenome
bull Many imprinted genes are involved in growth and metabolism
Culture Environment
bull There is growing evidence that the environment in which an embryo
develops can effect its metabolism epigenetic alterations and
developmental potential all aspects of environment important
bull Culture conditions have been shown in mouse models to impact
epigenetic patterns of the embryo and especially the placenta
bull Specifically culture at atmospheric (20) oxygen tension as compared to
physiologic (5) oxygen tension resulted in marked differences in global
gene expression in particular genes involved in cell growth and
maintenance relative to embryos developed in vivo
Donrsquot stress gametes or embryos Maintain homeostatic environment
Healthy adults from IVF should be
our end point
bull Embryos do have the ability to adapt to less than optimal culture
conditions however this comes at a cost culminating in incrementally
lower rates of blastocyst andor viability post implantation
bull Exposing preimplantation embryo to stressors that impair function of
mitochondria not only alters survival to the blastocyst and resultant
viability but subsequent fetal growth and health
Egg Retrieval
Once the follicular fluid has been
aspirated the eggs are removed
from the fluid in an IVF chamber
and placed into culture media
Eggs are retrieved trans-vaginally
with the use of a Ovum needle attached
to a vaginal ultrasound probe
Follicle Scan
eggs are within
the follicles
bull Number of follicles does not equal number
of eggs retrieved
bull Average egg number is around 8-10
bull Quality not quantity everyone is different
Egg Maturation
bull Only mature eggs will fertilise
Germinal Vesicle Metaphase I Metaphase II
Mature Egg
Maturation
Semen Parameters
SPERM CONCENTRATION
gt15 MILLION PER ML
Natural Variations
SPERM MOTILITY
gt50 Swimming forward
Different grades of motility
SPERM MORPHOLOGY
lt96 Abnormal forms considered Normal
Genetic
Lifestyle
ANTI-SPERM ANTIBODIES
Sperm Retrieval
bull Ejaculation ndash Most common - Motile sperm have to be separated from the non motile and abnormal
sperm using a two part gradient system and centrifugation
- Sperm are then washed with culture media and are ready
for insemination
If indicated
bull Retrograde Ejaculation
bull Sperm extraction
Males that may require intervention for sperm retrieval
Obstructive azoospermia
eg Vasectomy
Congenital Absence of Vas deference (CAV)
Non-obstructive azoospermia
eg Kleinfelters
Y-chromosome deletion
Sertoli cell only
Virtual Azoospermia
Depending on the aetiology sperm can be retrieved by
bull Percutaneous Epididymal Sperm Aspiration (PESA) (sperm stored in epididymus aspirated using needle)
bull Testicular Sperm Aspiration (TESA) (tubules in testis which produce sperm aspirated using needle)
bull Open Testicular Biopsy (incision made into scrotum exposing testis and tubules retrieved)
Can be performed under local anaesthetic unless
incision required then need general anaesthetic
Sperm Retrieval ndash Testicular biopsy
Seminiferous Tubules from Testicular Biopsy
Insemination
bull Depends on sperm quality or patient history (previous IVF cycles)
ndash Standard Insemination (IVF)
ndash Microinjection (ICSI)
Microinjection (ICSI)
In cases where semen quality is sub-optimal the
sperm are injected into the egg using a procedure
called Intra-Cytoplasmic Sperm Injection (ICSI)
A single sperm is immobilized and drawn into a fine pipette
for injection into the egg The egg is held steady using a
ldquoholdingrdquo pipette
The sperm is expelled into the cytoplasm of the egg and the
pipette is withdrawn from the egg
ICSI
Fertilisation
Fertilisation (Day 1)
Fertilised egg Unfertilised Abnormal
(2 Pronuclei Visible) (gt2 PN)
eg
- Standard Insemination more
than 1 sperm has entered
- ICSI ndash Second polar body
hasnrsquot
been extruded by egg as above
bull Pronuclei appear
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Laboratory Environment
bull Need homeostatic non-toxic nurturing environment
bull Warm laboratory to minimise temperature fluctuations
bull Assure nothing in lab is toxic
bull Keep lab free of VOCs ndash No cardboard in lab
ndash Air new equipment outside of lab
ndash Use VOC free paints
ndash Check VOCs using VOC measuring device
bull Take care with cleaning reagents within the laboratory
bull Embryologists contributing to environment ndash No perfume lotions make up
IVF Laboratory
bull Optimal culture conditions for optimal pregnancy results
bull Commitment to Single Embryo Transfer
bull Embryo culture
bull Cryopreservation of sperm eggs embryos ovarian tissue
bull Manage patient expectations through flexibility and choice
Function of the Embryology Laboratory
bull Culture media preparation
bull Egg collections (oocyte retrieval egg harvesting EPU VPU)
bull Sperm Preparation (and sometimes extraction)
bull Insemination
bull Embryo Culture
bull Embryo biopsy (as required)
bull Embryo transfer
bull Gameteembryo freezing and thawingvitrification and
warming
ndash With respect to our patients
ndash With the highest attention to detail
ndash With extensive witnessing procedures
Culture conditions and quality control bull Most common Complex Sequential Culture media used
ndash Complex means it provides all nutrients including amino acids and
protein for optimal embryo growth
ndash Sequential as it mimics the environment in the fallopian tubes then
uterus thus the culture media embryos are exposed to is changed at
appropriate times of development adapting to embryorsquos
requirements
bull Importance of temperature and pH
ndash Controlled environment essential for optimal
embryo growth therefore we use
bull IVF Chambers
bull Mincs
bull Strict QCQA Program
What influence does specific culture media and
environment have on our embryos
What is our end point in IVF
What is in culture media
bull Water
bull Salts
bull Energy substrates glucose pyruvate lactate
bull Buffer NaHCO3MOPs
bull Non-essential Amino Acids
bull Essential Amino Acids
bull Glutamine
bull Chelators ndash mainly EDTA
bull Macromolecules AlbuminHyaluronan
bull Antibiotics
bull Phenol Red
What is the purpose of culture media
Assist in preparing gametes for fertilisation and
grow embryos to then be placed into uterus
Differences between oviduct and uterus in mammalian embryos
Component Oviduct Uterus
Glucose concentration 05 mM 315 mM
Pyruvate concentration 032 mM 010 mM
Lactate concentration 105 mM 52 mM
Oxygen concentration 8 15
Carbon dioxide conc 12 10
pH 75 71
Glycine concentration 277 1933
Alanine concentration 05 124
Serine concentration 03 2080
Differences in the physiology of the mammalian embryo for
development from the zygote to the blastocyst stage
Precompaction stage Postcompaction stage
Low biosynthetic activity High biosynthetic activity
Low QO2 (Metabolic Quotient) High QO2 (Metabolic Quotient)
Pyruvate-based metabolism Glucose-based metabolism
Maternal genome Embryonic genome
Single cell Transporting epithelium
Low ability to maintain cellular Complex systems for maintenance
homeostasis of cellular homeostasis
Totipotent Differentiation into inner cell mass
and trophectoderm
Oviduct Uterus
uarr pyruvate darrpyruvate
uarrlactate darr lactate
darrglucose uarr glucose
uarrnon-ess AA uarr non-ess AA
uarr essential AA
Does culture media matter if your patients are
getting pregnant
bull Evidence that there are differences in DNA methylation patterns in
placental and umbilical blood samples from IVF children
compared to naturally conceived
bull There is an association with ART and specific imprinting disorders
bull SubfertilityInfertility hormone stimulation in vitro culture uterine
environment appear to influence the proper establishment and
maintenance of genomic imprints in the developing epigenome
Epigenetics amp Imprinting
bull Epigenetics Chromatin modifications that regulate gene activity
that are not due to DNA sequence changes (Saitou et al 2012)
bull Imprinted genes are genes whose expression is determined by
the parent that contributed them
bull Imprinted genes violate the usual rule of inheritance that both
alleles in a heterozygote are equally expressed
bull Genomic imprinting is a specialized epigenetic mechanism that
employs repressive modifications to silence one parental allele
while activating modifications on the other parental allele enable
expression Hirasawa amp Feil 2010
bull DNA methylation and histone modifications are two epigenetic
mechanisms that alter the functional state of chromatin activating
or repressing gene expression
In Vitro Embryo culture ndash Culture Media
bull Does culture media effect the health of the embryo
bull Suboptimal media may compromising imprint maintenance
ndash Mouse embryos cultured in deficient media displayed
biallelicH19 expression in postimplantation extraembryonic tissues
(Sasaki et al 1995)
ndash Whittens but not potassium simplex optimized medium plus amino acids
(KSOMaa) cultured embryos exhibited biallelic H19 expression
(Doherty et al 2000)
bull Extremely important for culture media to provide all the nutritional
requirements when required
bull Metabolites and cofactors are important regulators of the
epigenome
bull Many imprinted genes are involved in growth and metabolism
Culture Environment
bull There is growing evidence that the environment in which an embryo
develops can effect its metabolism epigenetic alterations and
developmental potential all aspects of environment important
bull Culture conditions have been shown in mouse models to impact
epigenetic patterns of the embryo and especially the placenta
bull Specifically culture at atmospheric (20) oxygen tension as compared to
physiologic (5) oxygen tension resulted in marked differences in global
gene expression in particular genes involved in cell growth and
maintenance relative to embryos developed in vivo
Donrsquot stress gametes or embryos Maintain homeostatic environment
Healthy adults from IVF should be
our end point
bull Embryos do have the ability to adapt to less than optimal culture
conditions however this comes at a cost culminating in incrementally
lower rates of blastocyst andor viability post implantation
bull Exposing preimplantation embryo to stressors that impair function of
mitochondria not only alters survival to the blastocyst and resultant
viability but subsequent fetal growth and health
Egg Retrieval
Once the follicular fluid has been
aspirated the eggs are removed
from the fluid in an IVF chamber
and placed into culture media
Eggs are retrieved trans-vaginally
with the use of a Ovum needle attached
to a vaginal ultrasound probe
Follicle Scan
eggs are within
the follicles
bull Number of follicles does not equal number
of eggs retrieved
bull Average egg number is around 8-10
bull Quality not quantity everyone is different
Egg Maturation
bull Only mature eggs will fertilise
Germinal Vesicle Metaphase I Metaphase II
Mature Egg
Maturation
Semen Parameters
SPERM CONCENTRATION
gt15 MILLION PER ML
Natural Variations
SPERM MOTILITY
gt50 Swimming forward
Different grades of motility
SPERM MORPHOLOGY
lt96 Abnormal forms considered Normal
Genetic
Lifestyle
ANTI-SPERM ANTIBODIES
Sperm Retrieval
bull Ejaculation ndash Most common - Motile sperm have to be separated from the non motile and abnormal
sperm using a two part gradient system and centrifugation
- Sperm are then washed with culture media and are ready
for insemination
If indicated
bull Retrograde Ejaculation
bull Sperm extraction
Males that may require intervention for sperm retrieval
Obstructive azoospermia
eg Vasectomy
Congenital Absence of Vas deference (CAV)
Non-obstructive azoospermia
eg Kleinfelters
Y-chromosome deletion
Sertoli cell only
Virtual Azoospermia
Depending on the aetiology sperm can be retrieved by
bull Percutaneous Epididymal Sperm Aspiration (PESA) (sperm stored in epididymus aspirated using needle)
bull Testicular Sperm Aspiration (TESA) (tubules in testis which produce sperm aspirated using needle)
bull Open Testicular Biopsy (incision made into scrotum exposing testis and tubules retrieved)
Can be performed under local anaesthetic unless
incision required then need general anaesthetic
Sperm Retrieval ndash Testicular biopsy
Seminiferous Tubules from Testicular Biopsy
Insemination
bull Depends on sperm quality or patient history (previous IVF cycles)
ndash Standard Insemination (IVF)
ndash Microinjection (ICSI)
Microinjection (ICSI)
In cases where semen quality is sub-optimal the
sperm are injected into the egg using a procedure
called Intra-Cytoplasmic Sperm Injection (ICSI)
A single sperm is immobilized and drawn into a fine pipette
for injection into the egg The egg is held steady using a
ldquoholdingrdquo pipette
The sperm is expelled into the cytoplasm of the egg and the
pipette is withdrawn from the egg
ICSI
Fertilisation
Fertilisation (Day 1)
Fertilised egg Unfertilised Abnormal
(2 Pronuclei Visible) (gt2 PN)
eg
- Standard Insemination more
than 1 sperm has entered
- ICSI ndash Second polar body
hasnrsquot
been extruded by egg as above
bull Pronuclei appear
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
IVF Laboratory
bull Optimal culture conditions for optimal pregnancy results
bull Commitment to Single Embryo Transfer
bull Embryo culture
bull Cryopreservation of sperm eggs embryos ovarian tissue
bull Manage patient expectations through flexibility and choice
Function of the Embryology Laboratory
bull Culture media preparation
bull Egg collections (oocyte retrieval egg harvesting EPU VPU)
bull Sperm Preparation (and sometimes extraction)
bull Insemination
bull Embryo Culture
bull Embryo biopsy (as required)
bull Embryo transfer
bull Gameteembryo freezing and thawingvitrification and
warming
ndash With respect to our patients
ndash With the highest attention to detail
ndash With extensive witnessing procedures
Culture conditions and quality control bull Most common Complex Sequential Culture media used
ndash Complex means it provides all nutrients including amino acids and
protein for optimal embryo growth
ndash Sequential as it mimics the environment in the fallopian tubes then
uterus thus the culture media embryos are exposed to is changed at
appropriate times of development adapting to embryorsquos
requirements
bull Importance of temperature and pH
ndash Controlled environment essential for optimal
embryo growth therefore we use
bull IVF Chambers
bull Mincs
bull Strict QCQA Program
What influence does specific culture media and
environment have on our embryos
What is our end point in IVF
What is in culture media
bull Water
bull Salts
bull Energy substrates glucose pyruvate lactate
bull Buffer NaHCO3MOPs
bull Non-essential Amino Acids
bull Essential Amino Acids
bull Glutamine
bull Chelators ndash mainly EDTA
bull Macromolecules AlbuminHyaluronan
bull Antibiotics
bull Phenol Red
What is the purpose of culture media
Assist in preparing gametes for fertilisation and
grow embryos to then be placed into uterus
Differences between oviduct and uterus in mammalian embryos
Component Oviduct Uterus
Glucose concentration 05 mM 315 mM
Pyruvate concentration 032 mM 010 mM
Lactate concentration 105 mM 52 mM
Oxygen concentration 8 15
Carbon dioxide conc 12 10
pH 75 71
Glycine concentration 277 1933
Alanine concentration 05 124
Serine concentration 03 2080
Differences in the physiology of the mammalian embryo for
development from the zygote to the blastocyst stage
Precompaction stage Postcompaction stage
Low biosynthetic activity High biosynthetic activity
Low QO2 (Metabolic Quotient) High QO2 (Metabolic Quotient)
Pyruvate-based metabolism Glucose-based metabolism
Maternal genome Embryonic genome
Single cell Transporting epithelium
Low ability to maintain cellular Complex systems for maintenance
homeostasis of cellular homeostasis
Totipotent Differentiation into inner cell mass
and trophectoderm
Oviduct Uterus
uarr pyruvate darrpyruvate
uarrlactate darr lactate
darrglucose uarr glucose
uarrnon-ess AA uarr non-ess AA
uarr essential AA
Does culture media matter if your patients are
getting pregnant
bull Evidence that there are differences in DNA methylation patterns in
placental and umbilical blood samples from IVF children
compared to naturally conceived
bull There is an association with ART and specific imprinting disorders
bull SubfertilityInfertility hormone stimulation in vitro culture uterine
environment appear to influence the proper establishment and
maintenance of genomic imprints in the developing epigenome
Epigenetics amp Imprinting
bull Epigenetics Chromatin modifications that regulate gene activity
that are not due to DNA sequence changes (Saitou et al 2012)
bull Imprinted genes are genes whose expression is determined by
the parent that contributed them
bull Imprinted genes violate the usual rule of inheritance that both
alleles in a heterozygote are equally expressed
bull Genomic imprinting is a specialized epigenetic mechanism that
employs repressive modifications to silence one parental allele
while activating modifications on the other parental allele enable
expression Hirasawa amp Feil 2010
bull DNA methylation and histone modifications are two epigenetic
mechanisms that alter the functional state of chromatin activating
or repressing gene expression
In Vitro Embryo culture ndash Culture Media
bull Does culture media effect the health of the embryo
bull Suboptimal media may compromising imprint maintenance
ndash Mouse embryos cultured in deficient media displayed
biallelicH19 expression in postimplantation extraembryonic tissues
(Sasaki et al 1995)
ndash Whittens but not potassium simplex optimized medium plus amino acids
(KSOMaa) cultured embryos exhibited biallelic H19 expression
(Doherty et al 2000)
bull Extremely important for culture media to provide all the nutritional
requirements when required
bull Metabolites and cofactors are important regulators of the
epigenome
bull Many imprinted genes are involved in growth and metabolism
Culture Environment
bull There is growing evidence that the environment in which an embryo
develops can effect its metabolism epigenetic alterations and
developmental potential all aspects of environment important
bull Culture conditions have been shown in mouse models to impact
epigenetic patterns of the embryo and especially the placenta
bull Specifically culture at atmospheric (20) oxygen tension as compared to
physiologic (5) oxygen tension resulted in marked differences in global
gene expression in particular genes involved in cell growth and
maintenance relative to embryos developed in vivo
Donrsquot stress gametes or embryos Maintain homeostatic environment
Healthy adults from IVF should be
our end point
bull Embryos do have the ability to adapt to less than optimal culture
conditions however this comes at a cost culminating in incrementally
lower rates of blastocyst andor viability post implantation
bull Exposing preimplantation embryo to stressors that impair function of
mitochondria not only alters survival to the blastocyst and resultant
viability but subsequent fetal growth and health
Egg Retrieval
Once the follicular fluid has been
aspirated the eggs are removed
from the fluid in an IVF chamber
and placed into culture media
Eggs are retrieved trans-vaginally
with the use of a Ovum needle attached
to a vaginal ultrasound probe
Follicle Scan
eggs are within
the follicles
bull Number of follicles does not equal number
of eggs retrieved
bull Average egg number is around 8-10
bull Quality not quantity everyone is different
Egg Maturation
bull Only mature eggs will fertilise
Germinal Vesicle Metaphase I Metaphase II
Mature Egg
Maturation
Semen Parameters
SPERM CONCENTRATION
gt15 MILLION PER ML
Natural Variations
SPERM MOTILITY
gt50 Swimming forward
Different grades of motility
SPERM MORPHOLOGY
lt96 Abnormal forms considered Normal
Genetic
Lifestyle
ANTI-SPERM ANTIBODIES
Sperm Retrieval
bull Ejaculation ndash Most common - Motile sperm have to be separated from the non motile and abnormal
sperm using a two part gradient system and centrifugation
- Sperm are then washed with culture media and are ready
for insemination
If indicated
bull Retrograde Ejaculation
bull Sperm extraction
Males that may require intervention for sperm retrieval
Obstructive azoospermia
eg Vasectomy
Congenital Absence of Vas deference (CAV)
Non-obstructive azoospermia
eg Kleinfelters
Y-chromosome deletion
Sertoli cell only
Virtual Azoospermia
Depending on the aetiology sperm can be retrieved by
bull Percutaneous Epididymal Sperm Aspiration (PESA) (sperm stored in epididymus aspirated using needle)
bull Testicular Sperm Aspiration (TESA) (tubules in testis which produce sperm aspirated using needle)
bull Open Testicular Biopsy (incision made into scrotum exposing testis and tubules retrieved)
Can be performed under local anaesthetic unless
incision required then need general anaesthetic
Sperm Retrieval ndash Testicular biopsy
Seminiferous Tubules from Testicular Biopsy
Insemination
bull Depends on sperm quality or patient history (previous IVF cycles)
ndash Standard Insemination (IVF)
ndash Microinjection (ICSI)
Microinjection (ICSI)
In cases where semen quality is sub-optimal the
sperm are injected into the egg using a procedure
called Intra-Cytoplasmic Sperm Injection (ICSI)
A single sperm is immobilized and drawn into a fine pipette
for injection into the egg The egg is held steady using a
ldquoholdingrdquo pipette
The sperm is expelled into the cytoplasm of the egg and the
pipette is withdrawn from the egg
ICSI
Fertilisation
Fertilisation (Day 1)
Fertilised egg Unfertilised Abnormal
(2 Pronuclei Visible) (gt2 PN)
eg
- Standard Insemination more
than 1 sperm has entered
- ICSI ndash Second polar body
hasnrsquot
been extruded by egg as above
bull Pronuclei appear
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Function of the Embryology Laboratory
bull Culture media preparation
bull Egg collections (oocyte retrieval egg harvesting EPU VPU)
bull Sperm Preparation (and sometimes extraction)
bull Insemination
bull Embryo Culture
bull Embryo biopsy (as required)
bull Embryo transfer
bull Gameteembryo freezing and thawingvitrification and
warming
ndash With respect to our patients
ndash With the highest attention to detail
ndash With extensive witnessing procedures
Culture conditions and quality control bull Most common Complex Sequential Culture media used
ndash Complex means it provides all nutrients including amino acids and
protein for optimal embryo growth
ndash Sequential as it mimics the environment in the fallopian tubes then
uterus thus the culture media embryos are exposed to is changed at
appropriate times of development adapting to embryorsquos
requirements
bull Importance of temperature and pH
ndash Controlled environment essential for optimal
embryo growth therefore we use
bull IVF Chambers
bull Mincs
bull Strict QCQA Program
What influence does specific culture media and
environment have on our embryos
What is our end point in IVF
What is in culture media
bull Water
bull Salts
bull Energy substrates glucose pyruvate lactate
bull Buffer NaHCO3MOPs
bull Non-essential Amino Acids
bull Essential Amino Acids
bull Glutamine
bull Chelators ndash mainly EDTA
bull Macromolecules AlbuminHyaluronan
bull Antibiotics
bull Phenol Red
What is the purpose of culture media
Assist in preparing gametes for fertilisation and
grow embryos to then be placed into uterus
Differences between oviduct and uterus in mammalian embryos
Component Oviduct Uterus
Glucose concentration 05 mM 315 mM
Pyruvate concentration 032 mM 010 mM
Lactate concentration 105 mM 52 mM
Oxygen concentration 8 15
Carbon dioxide conc 12 10
pH 75 71
Glycine concentration 277 1933
Alanine concentration 05 124
Serine concentration 03 2080
Differences in the physiology of the mammalian embryo for
development from the zygote to the blastocyst stage
Precompaction stage Postcompaction stage
Low biosynthetic activity High biosynthetic activity
Low QO2 (Metabolic Quotient) High QO2 (Metabolic Quotient)
Pyruvate-based metabolism Glucose-based metabolism
Maternal genome Embryonic genome
Single cell Transporting epithelium
Low ability to maintain cellular Complex systems for maintenance
homeostasis of cellular homeostasis
Totipotent Differentiation into inner cell mass
and trophectoderm
Oviduct Uterus
uarr pyruvate darrpyruvate
uarrlactate darr lactate
darrglucose uarr glucose
uarrnon-ess AA uarr non-ess AA
uarr essential AA
Does culture media matter if your patients are
getting pregnant
bull Evidence that there are differences in DNA methylation patterns in
placental and umbilical blood samples from IVF children
compared to naturally conceived
bull There is an association with ART and specific imprinting disorders
bull SubfertilityInfertility hormone stimulation in vitro culture uterine
environment appear to influence the proper establishment and
maintenance of genomic imprints in the developing epigenome
Epigenetics amp Imprinting
bull Epigenetics Chromatin modifications that regulate gene activity
that are not due to DNA sequence changes (Saitou et al 2012)
bull Imprinted genes are genes whose expression is determined by
the parent that contributed them
bull Imprinted genes violate the usual rule of inheritance that both
alleles in a heterozygote are equally expressed
bull Genomic imprinting is a specialized epigenetic mechanism that
employs repressive modifications to silence one parental allele
while activating modifications on the other parental allele enable
expression Hirasawa amp Feil 2010
bull DNA methylation and histone modifications are two epigenetic
mechanisms that alter the functional state of chromatin activating
or repressing gene expression
In Vitro Embryo culture ndash Culture Media
bull Does culture media effect the health of the embryo
bull Suboptimal media may compromising imprint maintenance
ndash Mouse embryos cultured in deficient media displayed
biallelicH19 expression in postimplantation extraembryonic tissues
(Sasaki et al 1995)
ndash Whittens but not potassium simplex optimized medium plus amino acids
(KSOMaa) cultured embryos exhibited biallelic H19 expression
(Doherty et al 2000)
bull Extremely important for culture media to provide all the nutritional
requirements when required
bull Metabolites and cofactors are important regulators of the
epigenome
bull Many imprinted genes are involved in growth and metabolism
Culture Environment
bull There is growing evidence that the environment in which an embryo
develops can effect its metabolism epigenetic alterations and
developmental potential all aspects of environment important
bull Culture conditions have been shown in mouse models to impact
epigenetic patterns of the embryo and especially the placenta
bull Specifically culture at atmospheric (20) oxygen tension as compared to
physiologic (5) oxygen tension resulted in marked differences in global
gene expression in particular genes involved in cell growth and
maintenance relative to embryos developed in vivo
Donrsquot stress gametes or embryos Maintain homeostatic environment
Healthy adults from IVF should be
our end point
bull Embryos do have the ability to adapt to less than optimal culture
conditions however this comes at a cost culminating in incrementally
lower rates of blastocyst andor viability post implantation
bull Exposing preimplantation embryo to stressors that impair function of
mitochondria not only alters survival to the blastocyst and resultant
viability but subsequent fetal growth and health
Egg Retrieval
Once the follicular fluid has been
aspirated the eggs are removed
from the fluid in an IVF chamber
and placed into culture media
Eggs are retrieved trans-vaginally
with the use of a Ovum needle attached
to a vaginal ultrasound probe
Follicle Scan
eggs are within
the follicles
bull Number of follicles does not equal number
of eggs retrieved
bull Average egg number is around 8-10
bull Quality not quantity everyone is different
Egg Maturation
bull Only mature eggs will fertilise
Germinal Vesicle Metaphase I Metaphase II
Mature Egg
Maturation
Semen Parameters
SPERM CONCENTRATION
gt15 MILLION PER ML
Natural Variations
SPERM MOTILITY
gt50 Swimming forward
Different grades of motility
SPERM MORPHOLOGY
lt96 Abnormal forms considered Normal
Genetic
Lifestyle
ANTI-SPERM ANTIBODIES
Sperm Retrieval
bull Ejaculation ndash Most common - Motile sperm have to be separated from the non motile and abnormal
sperm using a two part gradient system and centrifugation
- Sperm are then washed with culture media and are ready
for insemination
If indicated
bull Retrograde Ejaculation
bull Sperm extraction
Males that may require intervention for sperm retrieval
Obstructive azoospermia
eg Vasectomy
Congenital Absence of Vas deference (CAV)
Non-obstructive azoospermia
eg Kleinfelters
Y-chromosome deletion
Sertoli cell only
Virtual Azoospermia
Depending on the aetiology sperm can be retrieved by
bull Percutaneous Epididymal Sperm Aspiration (PESA) (sperm stored in epididymus aspirated using needle)
bull Testicular Sperm Aspiration (TESA) (tubules in testis which produce sperm aspirated using needle)
bull Open Testicular Biopsy (incision made into scrotum exposing testis and tubules retrieved)
Can be performed under local anaesthetic unless
incision required then need general anaesthetic
Sperm Retrieval ndash Testicular biopsy
Seminiferous Tubules from Testicular Biopsy
Insemination
bull Depends on sperm quality or patient history (previous IVF cycles)
ndash Standard Insemination (IVF)
ndash Microinjection (ICSI)
Microinjection (ICSI)
In cases where semen quality is sub-optimal the
sperm are injected into the egg using a procedure
called Intra-Cytoplasmic Sperm Injection (ICSI)
A single sperm is immobilized and drawn into a fine pipette
for injection into the egg The egg is held steady using a
ldquoholdingrdquo pipette
The sperm is expelled into the cytoplasm of the egg and the
pipette is withdrawn from the egg
ICSI
Fertilisation
Fertilisation (Day 1)
Fertilised egg Unfertilised Abnormal
(2 Pronuclei Visible) (gt2 PN)
eg
- Standard Insemination more
than 1 sperm has entered
- ICSI ndash Second polar body
hasnrsquot
been extruded by egg as above
bull Pronuclei appear
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Culture conditions and quality control bull Most common Complex Sequential Culture media used
ndash Complex means it provides all nutrients including amino acids and
protein for optimal embryo growth
ndash Sequential as it mimics the environment in the fallopian tubes then
uterus thus the culture media embryos are exposed to is changed at
appropriate times of development adapting to embryorsquos
requirements
bull Importance of temperature and pH
ndash Controlled environment essential for optimal
embryo growth therefore we use
bull IVF Chambers
bull Mincs
bull Strict QCQA Program
What influence does specific culture media and
environment have on our embryos
What is our end point in IVF
What is in culture media
bull Water
bull Salts
bull Energy substrates glucose pyruvate lactate
bull Buffer NaHCO3MOPs
bull Non-essential Amino Acids
bull Essential Amino Acids
bull Glutamine
bull Chelators ndash mainly EDTA
bull Macromolecules AlbuminHyaluronan
bull Antibiotics
bull Phenol Red
What is the purpose of culture media
Assist in preparing gametes for fertilisation and
grow embryos to then be placed into uterus
Differences between oviduct and uterus in mammalian embryos
Component Oviduct Uterus
Glucose concentration 05 mM 315 mM
Pyruvate concentration 032 mM 010 mM
Lactate concentration 105 mM 52 mM
Oxygen concentration 8 15
Carbon dioxide conc 12 10
pH 75 71
Glycine concentration 277 1933
Alanine concentration 05 124
Serine concentration 03 2080
Differences in the physiology of the mammalian embryo for
development from the zygote to the blastocyst stage
Precompaction stage Postcompaction stage
Low biosynthetic activity High biosynthetic activity
Low QO2 (Metabolic Quotient) High QO2 (Metabolic Quotient)
Pyruvate-based metabolism Glucose-based metabolism
Maternal genome Embryonic genome
Single cell Transporting epithelium
Low ability to maintain cellular Complex systems for maintenance
homeostasis of cellular homeostasis
Totipotent Differentiation into inner cell mass
and trophectoderm
Oviduct Uterus
uarr pyruvate darrpyruvate
uarrlactate darr lactate
darrglucose uarr glucose
uarrnon-ess AA uarr non-ess AA
uarr essential AA
Does culture media matter if your patients are
getting pregnant
bull Evidence that there are differences in DNA methylation patterns in
placental and umbilical blood samples from IVF children
compared to naturally conceived
bull There is an association with ART and specific imprinting disorders
bull SubfertilityInfertility hormone stimulation in vitro culture uterine
environment appear to influence the proper establishment and
maintenance of genomic imprints in the developing epigenome
Epigenetics amp Imprinting
bull Epigenetics Chromatin modifications that regulate gene activity
that are not due to DNA sequence changes (Saitou et al 2012)
bull Imprinted genes are genes whose expression is determined by
the parent that contributed them
bull Imprinted genes violate the usual rule of inheritance that both
alleles in a heterozygote are equally expressed
bull Genomic imprinting is a specialized epigenetic mechanism that
employs repressive modifications to silence one parental allele
while activating modifications on the other parental allele enable
expression Hirasawa amp Feil 2010
bull DNA methylation and histone modifications are two epigenetic
mechanisms that alter the functional state of chromatin activating
or repressing gene expression
In Vitro Embryo culture ndash Culture Media
bull Does culture media effect the health of the embryo
bull Suboptimal media may compromising imprint maintenance
ndash Mouse embryos cultured in deficient media displayed
biallelicH19 expression in postimplantation extraembryonic tissues
(Sasaki et al 1995)
ndash Whittens but not potassium simplex optimized medium plus amino acids
(KSOMaa) cultured embryos exhibited biallelic H19 expression
(Doherty et al 2000)
bull Extremely important for culture media to provide all the nutritional
requirements when required
bull Metabolites and cofactors are important regulators of the
epigenome
bull Many imprinted genes are involved in growth and metabolism
Culture Environment
bull There is growing evidence that the environment in which an embryo
develops can effect its metabolism epigenetic alterations and
developmental potential all aspects of environment important
bull Culture conditions have been shown in mouse models to impact
epigenetic patterns of the embryo and especially the placenta
bull Specifically culture at atmospheric (20) oxygen tension as compared to
physiologic (5) oxygen tension resulted in marked differences in global
gene expression in particular genes involved in cell growth and
maintenance relative to embryos developed in vivo
Donrsquot stress gametes or embryos Maintain homeostatic environment
Healthy adults from IVF should be
our end point
bull Embryos do have the ability to adapt to less than optimal culture
conditions however this comes at a cost culminating in incrementally
lower rates of blastocyst andor viability post implantation
bull Exposing preimplantation embryo to stressors that impair function of
mitochondria not only alters survival to the blastocyst and resultant
viability but subsequent fetal growth and health
Egg Retrieval
Once the follicular fluid has been
aspirated the eggs are removed
from the fluid in an IVF chamber
and placed into culture media
Eggs are retrieved trans-vaginally
with the use of a Ovum needle attached
to a vaginal ultrasound probe
Follicle Scan
eggs are within
the follicles
bull Number of follicles does not equal number
of eggs retrieved
bull Average egg number is around 8-10
bull Quality not quantity everyone is different
Egg Maturation
bull Only mature eggs will fertilise
Germinal Vesicle Metaphase I Metaphase II
Mature Egg
Maturation
Semen Parameters
SPERM CONCENTRATION
gt15 MILLION PER ML
Natural Variations
SPERM MOTILITY
gt50 Swimming forward
Different grades of motility
SPERM MORPHOLOGY
lt96 Abnormal forms considered Normal
Genetic
Lifestyle
ANTI-SPERM ANTIBODIES
Sperm Retrieval
bull Ejaculation ndash Most common - Motile sperm have to be separated from the non motile and abnormal
sperm using a two part gradient system and centrifugation
- Sperm are then washed with culture media and are ready
for insemination
If indicated
bull Retrograde Ejaculation
bull Sperm extraction
Males that may require intervention for sperm retrieval
Obstructive azoospermia
eg Vasectomy
Congenital Absence of Vas deference (CAV)
Non-obstructive azoospermia
eg Kleinfelters
Y-chromosome deletion
Sertoli cell only
Virtual Azoospermia
Depending on the aetiology sperm can be retrieved by
bull Percutaneous Epididymal Sperm Aspiration (PESA) (sperm stored in epididymus aspirated using needle)
bull Testicular Sperm Aspiration (TESA) (tubules in testis which produce sperm aspirated using needle)
bull Open Testicular Biopsy (incision made into scrotum exposing testis and tubules retrieved)
Can be performed under local anaesthetic unless
incision required then need general anaesthetic
Sperm Retrieval ndash Testicular biopsy
Seminiferous Tubules from Testicular Biopsy
Insemination
bull Depends on sperm quality or patient history (previous IVF cycles)
ndash Standard Insemination (IVF)
ndash Microinjection (ICSI)
Microinjection (ICSI)
In cases where semen quality is sub-optimal the
sperm are injected into the egg using a procedure
called Intra-Cytoplasmic Sperm Injection (ICSI)
A single sperm is immobilized and drawn into a fine pipette
for injection into the egg The egg is held steady using a
ldquoholdingrdquo pipette
The sperm is expelled into the cytoplasm of the egg and the
pipette is withdrawn from the egg
ICSI
Fertilisation
Fertilisation (Day 1)
Fertilised egg Unfertilised Abnormal
(2 Pronuclei Visible) (gt2 PN)
eg
- Standard Insemination more
than 1 sperm has entered
- ICSI ndash Second polar body
hasnrsquot
been extruded by egg as above
bull Pronuclei appear
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
What influence does specific culture media and
environment have on our embryos
What is our end point in IVF
What is in culture media
bull Water
bull Salts
bull Energy substrates glucose pyruvate lactate
bull Buffer NaHCO3MOPs
bull Non-essential Amino Acids
bull Essential Amino Acids
bull Glutamine
bull Chelators ndash mainly EDTA
bull Macromolecules AlbuminHyaluronan
bull Antibiotics
bull Phenol Red
What is the purpose of culture media
Assist in preparing gametes for fertilisation and
grow embryos to then be placed into uterus
Differences between oviduct and uterus in mammalian embryos
Component Oviduct Uterus
Glucose concentration 05 mM 315 mM
Pyruvate concentration 032 mM 010 mM
Lactate concentration 105 mM 52 mM
Oxygen concentration 8 15
Carbon dioxide conc 12 10
pH 75 71
Glycine concentration 277 1933
Alanine concentration 05 124
Serine concentration 03 2080
Differences in the physiology of the mammalian embryo for
development from the zygote to the blastocyst stage
Precompaction stage Postcompaction stage
Low biosynthetic activity High biosynthetic activity
Low QO2 (Metabolic Quotient) High QO2 (Metabolic Quotient)
Pyruvate-based metabolism Glucose-based metabolism
Maternal genome Embryonic genome
Single cell Transporting epithelium
Low ability to maintain cellular Complex systems for maintenance
homeostasis of cellular homeostasis
Totipotent Differentiation into inner cell mass
and trophectoderm
Oviduct Uterus
uarr pyruvate darrpyruvate
uarrlactate darr lactate
darrglucose uarr glucose
uarrnon-ess AA uarr non-ess AA
uarr essential AA
Does culture media matter if your patients are
getting pregnant
bull Evidence that there are differences in DNA methylation patterns in
placental and umbilical blood samples from IVF children
compared to naturally conceived
bull There is an association with ART and specific imprinting disorders
bull SubfertilityInfertility hormone stimulation in vitro culture uterine
environment appear to influence the proper establishment and
maintenance of genomic imprints in the developing epigenome
Epigenetics amp Imprinting
bull Epigenetics Chromatin modifications that regulate gene activity
that are not due to DNA sequence changes (Saitou et al 2012)
bull Imprinted genes are genes whose expression is determined by
the parent that contributed them
bull Imprinted genes violate the usual rule of inheritance that both
alleles in a heterozygote are equally expressed
bull Genomic imprinting is a specialized epigenetic mechanism that
employs repressive modifications to silence one parental allele
while activating modifications on the other parental allele enable
expression Hirasawa amp Feil 2010
bull DNA methylation and histone modifications are two epigenetic
mechanisms that alter the functional state of chromatin activating
or repressing gene expression
In Vitro Embryo culture ndash Culture Media
bull Does culture media effect the health of the embryo
bull Suboptimal media may compromising imprint maintenance
ndash Mouse embryos cultured in deficient media displayed
biallelicH19 expression in postimplantation extraembryonic tissues
(Sasaki et al 1995)
ndash Whittens but not potassium simplex optimized medium plus amino acids
(KSOMaa) cultured embryos exhibited biallelic H19 expression
(Doherty et al 2000)
bull Extremely important for culture media to provide all the nutritional
requirements when required
bull Metabolites and cofactors are important regulators of the
epigenome
bull Many imprinted genes are involved in growth and metabolism
Culture Environment
bull There is growing evidence that the environment in which an embryo
develops can effect its metabolism epigenetic alterations and
developmental potential all aspects of environment important
bull Culture conditions have been shown in mouse models to impact
epigenetic patterns of the embryo and especially the placenta
bull Specifically culture at atmospheric (20) oxygen tension as compared to
physiologic (5) oxygen tension resulted in marked differences in global
gene expression in particular genes involved in cell growth and
maintenance relative to embryos developed in vivo
Donrsquot stress gametes or embryos Maintain homeostatic environment
Healthy adults from IVF should be
our end point
bull Embryos do have the ability to adapt to less than optimal culture
conditions however this comes at a cost culminating in incrementally
lower rates of blastocyst andor viability post implantation
bull Exposing preimplantation embryo to stressors that impair function of
mitochondria not only alters survival to the blastocyst and resultant
viability but subsequent fetal growth and health
Egg Retrieval
Once the follicular fluid has been
aspirated the eggs are removed
from the fluid in an IVF chamber
and placed into culture media
Eggs are retrieved trans-vaginally
with the use of a Ovum needle attached
to a vaginal ultrasound probe
Follicle Scan
eggs are within
the follicles
bull Number of follicles does not equal number
of eggs retrieved
bull Average egg number is around 8-10
bull Quality not quantity everyone is different
Egg Maturation
bull Only mature eggs will fertilise
Germinal Vesicle Metaphase I Metaphase II
Mature Egg
Maturation
Semen Parameters
SPERM CONCENTRATION
gt15 MILLION PER ML
Natural Variations
SPERM MOTILITY
gt50 Swimming forward
Different grades of motility
SPERM MORPHOLOGY
lt96 Abnormal forms considered Normal
Genetic
Lifestyle
ANTI-SPERM ANTIBODIES
Sperm Retrieval
bull Ejaculation ndash Most common - Motile sperm have to be separated from the non motile and abnormal
sperm using a two part gradient system and centrifugation
- Sperm are then washed with culture media and are ready
for insemination
If indicated
bull Retrograde Ejaculation
bull Sperm extraction
Males that may require intervention for sperm retrieval
Obstructive azoospermia
eg Vasectomy
Congenital Absence of Vas deference (CAV)
Non-obstructive azoospermia
eg Kleinfelters
Y-chromosome deletion
Sertoli cell only
Virtual Azoospermia
Depending on the aetiology sperm can be retrieved by
bull Percutaneous Epididymal Sperm Aspiration (PESA) (sperm stored in epididymus aspirated using needle)
bull Testicular Sperm Aspiration (TESA) (tubules in testis which produce sperm aspirated using needle)
bull Open Testicular Biopsy (incision made into scrotum exposing testis and tubules retrieved)
Can be performed under local anaesthetic unless
incision required then need general anaesthetic
Sperm Retrieval ndash Testicular biopsy
Seminiferous Tubules from Testicular Biopsy
Insemination
bull Depends on sperm quality or patient history (previous IVF cycles)
ndash Standard Insemination (IVF)
ndash Microinjection (ICSI)
Microinjection (ICSI)
In cases where semen quality is sub-optimal the
sperm are injected into the egg using a procedure
called Intra-Cytoplasmic Sperm Injection (ICSI)
A single sperm is immobilized and drawn into a fine pipette
for injection into the egg The egg is held steady using a
ldquoholdingrdquo pipette
The sperm is expelled into the cytoplasm of the egg and the
pipette is withdrawn from the egg
ICSI
Fertilisation
Fertilisation (Day 1)
Fertilised egg Unfertilised Abnormal
(2 Pronuclei Visible) (gt2 PN)
eg
- Standard Insemination more
than 1 sperm has entered
- ICSI ndash Second polar body
hasnrsquot
been extruded by egg as above
bull Pronuclei appear
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
What is in culture media
bull Water
bull Salts
bull Energy substrates glucose pyruvate lactate
bull Buffer NaHCO3MOPs
bull Non-essential Amino Acids
bull Essential Amino Acids
bull Glutamine
bull Chelators ndash mainly EDTA
bull Macromolecules AlbuminHyaluronan
bull Antibiotics
bull Phenol Red
What is the purpose of culture media
Assist in preparing gametes for fertilisation and
grow embryos to then be placed into uterus
Differences between oviduct and uterus in mammalian embryos
Component Oviduct Uterus
Glucose concentration 05 mM 315 mM
Pyruvate concentration 032 mM 010 mM
Lactate concentration 105 mM 52 mM
Oxygen concentration 8 15
Carbon dioxide conc 12 10
pH 75 71
Glycine concentration 277 1933
Alanine concentration 05 124
Serine concentration 03 2080
Differences in the physiology of the mammalian embryo for
development from the zygote to the blastocyst stage
Precompaction stage Postcompaction stage
Low biosynthetic activity High biosynthetic activity
Low QO2 (Metabolic Quotient) High QO2 (Metabolic Quotient)
Pyruvate-based metabolism Glucose-based metabolism
Maternal genome Embryonic genome
Single cell Transporting epithelium
Low ability to maintain cellular Complex systems for maintenance
homeostasis of cellular homeostasis
Totipotent Differentiation into inner cell mass
and trophectoderm
Oviduct Uterus
uarr pyruvate darrpyruvate
uarrlactate darr lactate
darrglucose uarr glucose
uarrnon-ess AA uarr non-ess AA
uarr essential AA
Does culture media matter if your patients are
getting pregnant
bull Evidence that there are differences in DNA methylation patterns in
placental and umbilical blood samples from IVF children
compared to naturally conceived
bull There is an association with ART and specific imprinting disorders
bull SubfertilityInfertility hormone stimulation in vitro culture uterine
environment appear to influence the proper establishment and
maintenance of genomic imprints in the developing epigenome
Epigenetics amp Imprinting
bull Epigenetics Chromatin modifications that regulate gene activity
that are not due to DNA sequence changes (Saitou et al 2012)
bull Imprinted genes are genes whose expression is determined by
the parent that contributed them
bull Imprinted genes violate the usual rule of inheritance that both
alleles in a heterozygote are equally expressed
bull Genomic imprinting is a specialized epigenetic mechanism that
employs repressive modifications to silence one parental allele
while activating modifications on the other parental allele enable
expression Hirasawa amp Feil 2010
bull DNA methylation and histone modifications are two epigenetic
mechanisms that alter the functional state of chromatin activating
or repressing gene expression
In Vitro Embryo culture ndash Culture Media
bull Does culture media effect the health of the embryo
bull Suboptimal media may compromising imprint maintenance
ndash Mouse embryos cultured in deficient media displayed
biallelicH19 expression in postimplantation extraembryonic tissues
(Sasaki et al 1995)
ndash Whittens but not potassium simplex optimized medium plus amino acids
(KSOMaa) cultured embryos exhibited biallelic H19 expression
(Doherty et al 2000)
bull Extremely important for culture media to provide all the nutritional
requirements when required
bull Metabolites and cofactors are important regulators of the
epigenome
bull Many imprinted genes are involved in growth and metabolism
Culture Environment
bull There is growing evidence that the environment in which an embryo
develops can effect its metabolism epigenetic alterations and
developmental potential all aspects of environment important
bull Culture conditions have been shown in mouse models to impact
epigenetic patterns of the embryo and especially the placenta
bull Specifically culture at atmospheric (20) oxygen tension as compared to
physiologic (5) oxygen tension resulted in marked differences in global
gene expression in particular genes involved in cell growth and
maintenance relative to embryos developed in vivo
Donrsquot stress gametes or embryos Maintain homeostatic environment
Healthy adults from IVF should be
our end point
bull Embryos do have the ability to adapt to less than optimal culture
conditions however this comes at a cost culminating in incrementally
lower rates of blastocyst andor viability post implantation
bull Exposing preimplantation embryo to stressors that impair function of
mitochondria not only alters survival to the blastocyst and resultant
viability but subsequent fetal growth and health
Egg Retrieval
Once the follicular fluid has been
aspirated the eggs are removed
from the fluid in an IVF chamber
and placed into culture media
Eggs are retrieved trans-vaginally
with the use of a Ovum needle attached
to a vaginal ultrasound probe
Follicle Scan
eggs are within
the follicles
bull Number of follicles does not equal number
of eggs retrieved
bull Average egg number is around 8-10
bull Quality not quantity everyone is different
Egg Maturation
bull Only mature eggs will fertilise
Germinal Vesicle Metaphase I Metaphase II
Mature Egg
Maturation
Semen Parameters
SPERM CONCENTRATION
gt15 MILLION PER ML
Natural Variations
SPERM MOTILITY
gt50 Swimming forward
Different grades of motility
SPERM MORPHOLOGY
lt96 Abnormal forms considered Normal
Genetic
Lifestyle
ANTI-SPERM ANTIBODIES
Sperm Retrieval
bull Ejaculation ndash Most common - Motile sperm have to be separated from the non motile and abnormal
sperm using a two part gradient system and centrifugation
- Sperm are then washed with culture media and are ready
for insemination
If indicated
bull Retrograde Ejaculation
bull Sperm extraction
Males that may require intervention for sperm retrieval
Obstructive azoospermia
eg Vasectomy
Congenital Absence of Vas deference (CAV)
Non-obstructive azoospermia
eg Kleinfelters
Y-chromosome deletion
Sertoli cell only
Virtual Azoospermia
Depending on the aetiology sperm can be retrieved by
bull Percutaneous Epididymal Sperm Aspiration (PESA) (sperm stored in epididymus aspirated using needle)
bull Testicular Sperm Aspiration (TESA) (tubules in testis which produce sperm aspirated using needle)
bull Open Testicular Biopsy (incision made into scrotum exposing testis and tubules retrieved)
Can be performed under local anaesthetic unless
incision required then need general anaesthetic
Sperm Retrieval ndash Testicular biopsy
Seminiferous Tubules from Testicular Biopsy
Insemination
bull Depends on sperm quality or patient history (previous IVF cycles)
ndash Standard Insemination (IVF)
ndash Microinjection (ICSI)
Microinjection (ICSI)
In cases where semen quality is sub-optimal the
sperm are injected into the egg using a procedure
called Intra-Cytoplasmic Sperm Injection (ICSI)
A single sperm is immobilized and drawn into a fine pipette
for injection into the egg The egg is held steady using a
ldquoholdingrdquo pipette
The sperm is expelled into the cytoplasm of the egg and the
pipette is withdrawn from the egg
ICSI
Fertilisation
Fertilisation (Day 1)
Fertilised egg Unfertilised Abnormal
(2 Pronuclei Visible) (gt2 PN)
eg
- Standard Insemination more
than 1 sperm has entered
- ICSI ndash Second polar body
hasnrsquot
been extruded by egg as above
bull Pronuclei appear
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
What is the purpose of culture media
Assist in preparing gametes for fertilisation and
grow embryos to then be placed into uterus
Differences between oviduct and uterus in mammalian embryos
Component Oviduct Uterus
Glucose concentration 05 mM 315 mM
Pyruvate concentration 032 mM 010 mM
Lactate concentration 105 mM 52 mM
Oxygen concentration 8 15
Carbon dioxide conc 12 10
pH 75 71
Glycine concentration 277 1933
Alanine concentration 05 124
Serine concentration 03 2080
Differences in the physiology of the mammalian embryo for
development from the zygote to the blastocyst stage
Precompaction stage Postcompaction stage
Low biosynthetic activity High biosynthetic activity
Low QO2 (Metabolic Quotient) High QO2 (Metabolic Quotient)
Pyruvate-based metabolism Glucose-based metabolism
Maternal genome Embryonic genome
Single cell Transporting epithelium
Low ability to maintain cellular Complex systems for maintenance
homeostasis of cellular homeostasis
Totipotent Differentiation into inner cell mass
and trophectoderm
Oviduct Uterus
uarr pyruvate darrpyruvate
uarrlactate darr lactate
darrglucose uarr glucose
uarrnon-ess AA uarr non-ess AA
uarr essential AA
Does culture media matter if your patients are
getting pregnant
bull Evidence that there are differences in DNA methylation patterns in
placental and umbilical blood samples from IVF children
compared to naturally conceived
bull There is an association with ART and specific imprinting disorders
bull SubfertilityInfertility hormone stimulation in vitro culture uterine
environment appear to influence the proper establishment and
maintenance of genomic imprints in the developing epigenome
Epigenetics amp Imprinting
bull Epigenetics Chromatin modifications that regulate gene activity
that are not due to DNA sequence changes (Saitou et al 2012)
bull Imprinted genes are genes whose expression is determined by
the parent that contributed them
bull Imprinted genes violate the usual rule of inheritance that both
alleles in a heterozygote are equally expressed
bull Genomic imprinting is a specialized epigenetic mechanism that
employs repressive modifications to silence one parental allele
while activating modifications on the other parental allele enable
expression Hirasawa amp Feil 2010
bull DNA methylation and histone modifications are two epigenetic
mechanisms that alter the functional state of chromatin activating
or repressing gene expression
In Vitro Embryo culture ndash Culture Media
bull Does culture media effect the health of the embryo
bull Suboptimal media may compromising imprint maintenance
ndash Mouse embryos cultured in deficient media displayed
biallelicH19 expression in postimplantation extraembryonic tissues
(Sasaki et al 1995)
ndash Whittens but not potassium simplex optimized medium plus amino acids
(KSOMaa) cultured embryos exhibited biallelic H19 expression
(Doherty et al 2000)
bull Extremely important for culture media to provide all the nutritional
requirements when required
bull Metabolites and cofactors are important regulators of the
epigenome
bull Many imprinted genes are involved in growth and metabolism
Culture Environment
bull There is growing evidence that the environment in which an embryo
develops can effect its metabolism epigenetic alterations and
developmental potential all aspects of environment important
bull Culture conditions have been shown in mouse models to impact
epigenetic patterns of the embryo and especially the placenta
bull Specifically culture at atmospheric (20) oxygen tension as compared to
physiologic (5) oxygen tension resulted in marked differences in global
gene expression in particular genes involved in cell growth and
maintenance relative to embryos developed in vivo
Donrsquot stress gametes or embryos Maintain homeostatic environment
Healthy adults from IVF should be
our end point
bull Embryos do have the ability to adapt to less than optimal culture
conditions however this comes at a cost culminating in incrementally
lower rates of blastocyst andor viability post implantation
bull Exposing preimplantation embryo to stressors that impair function of
mitochondria not only alters survival to the blastocyst and resultant
viability but subsequent fetal growth and health
Egg Retrieval
Once the follicular fluid has been
aspirated the eggs are removed
from the fluid in an IVF chamber
and placed into culture media
Eggs are retrieved trans-vaginally
with the use of a Ovum needle attached
to a vaginal ultrasound probe
Follicle Scan
eggs are within
the follicles
bull Number of follicles does not equal number
of eggs retrieved
bull Average egg number is around 8-10
bull Quality not quantity everyone is different
Egg Maturation
bull Only mature eggs will fertilise
Germinal Vesicle Metaphase I Metaphase II
Mature Egg
Maturation
Semen Parameters
SPERM CONCENTRATION
gt15 MILLION PER ML
Natural Variations
SPERM MOTILITY
gt50 Swimming forward
Different grades of motility
SPERM MORPHOLOGY
lt96 Abnormal forms considered Normal
Genetic
Lifestyle
ANTI-SPERM ANTIBODIES
Sperm Retrieval
bull Ejaculation ndash Most common - Motile sperm have to be separated from the non motile and abnormal
sperm using a two part gradient system and centrifugation
- Sperm are then washed with culture media and are ready
for insemination
If indicated
bull Retrograde Ejaculation
bull Sperm extraction
Males that may require intervention for sperm retrieval
Obstructive azoospermia
eg Vasectomy
Congenital Absence of Vas deference (CAV)
Non-obstructive azoospermia
eg Kleinfelters
Y-chromosome deletion
Sertoli cell only
Virtual Azoospermia
Depending on the aetiology sperm can be retrieved by
bull Percutaneous Epididymal Sperm Aspiration (PESA) (sperm stored in epididymus aspirated using needle)
bull Testicular Sperm Aspiration (TESA) (tubules in testis which produce sperm aspirated using needle)
bull Open Testicular Biopsy (incision made into scrotum exposing testis and tubules retrieved)
Can be performed under local anaesthetic unless
incision required then need general anaesthetic
Sperm Retrieval ndash Testicular biopsy
Seminiferous Tubules from Testicular Biopsy
Insemination
bull Depends on sperm quality or patient history (previous IVF cycles)
ndash Standard Insemination (IVF)
ndash Microinjection (ICSI)
Microinjection (ICSI)
In cases where semen quality is sub-optimal the
sperm are injected into the egg using a procedure
called Intra-Cytoplasmic Sperm Injection (ICSI)
A single sperm is immobilized and drawn into a fine pipette
for injection into the egg The egg is held steady using a
ldquoholdingrdquo pipette
The sperm is expelled into the cytoplasm of the egg and the
pipette is withdrawn from the egg
ICSI
Fertilisation
Fertilisation (Day 1)
Fertilised egg Unfertilised Abnormal
(2 Pronuclei Visible) (gt2 PN)
eg
- Standard Insemination more
than 1 sperm has entered
- ICSI ndash Second polar body
hasnrsquot
been extruded by egg as above
bull Pronuclei appear
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Differences between oviduct and uterus in mammalian embryos
Component Oviduct Uterus
Glucose concentration 05 mM 315 mM
Pyruvate concentration 032 mM 010 mM
Lactate concentration 105 mM 52 mM
Oxygen concentration 8 15
Carbon dioxide conc 12 10
pH 75 71
Glycine concentration 277 1933
Alanine concentration 05 124
Serine concentration 03 2080
Differences in the physiology of the mammalian embryo for
development from the zygote to the blastocyst stage
Precompaction stage Postcompaction stage
Low biosynthetic activity High biosynthetic activity
Low QO2 (Metabolic Quotient) High QO2 (Metabolic Quotient)
Pyruvate-based metabolism Glucose-based metabolism
Maternal genome Embryonic genome
Single cell Transporting epithelium
Low ability to maintain cellular Complex systems for maintenance
homeostasis of cellular homeostasis
Totipotent Differentiation into inner cell mass
and trophectoderm
Oviduct Uterus
uarr pyruvate darrpyruvate
uarrlactate darr lactate
darrglucose uarr glucose
uarrnon-ess AA uarr non-ess AA
uarr essential AA
Does culture media matter if your patients are
getting pregnant
bull Evidence that there are differences in DNA methylation patterns in
placental and umbilical blood samples from IVF children
compared to naturally conceived
bull There is an association with ART and specific imprinting disorders
bull SubfertilityInfertility hormone stimulation in vitro culture uterine
environment appear to influence the proper establishment and
maintenance of genomic imprints in the developing epigenome
Epigenetics amp Imprinting
bull Epigenetics Chromatin modifications that regulate gene activity
that are not due to DNA sequence changes (Saitou et al 2012)
bull Imprinted genes are genes whose expression is determined by
the parent that contributed them
bull Imprinted genes violate the usual rule of inheritance that both
alleles in a heterozygote are equally expressed
bull Genomic imprinting is a specialized epigenetic mechanism that
employs repressive modifications to silence one parental allele
while activating modifications on the other parental allele enable
expression Hirasawa amp Feil 2010
bull DNA methylation and histone modifications are two epigenetic
mechanisms that alter the functional state of chromatin activating
or repressing gene expression
In Vitro Embryo culture ndash Culture Media
bull Does culture media effect the health of the embryo
bull Suboptimal media may compromising imprint maintenance
ndash Mouse embryos cultured in deficient media displayed
biallelicH19 expression in postimplantation extraembryonic tissues
(Sasaki et al 1995)
ndash Whittens but not potassium simplex optimized medium plus amino acids
(KSOMaa) cultured embryos exhibited biallelic H19 expression
(Doherty et al 2000)
bull Extremely important for culture media to provide all the nutritional
requirements when required
bull Metabolites and cofactors are important regulators of the
epigenome
bull Many imprinted genes are involved in growth and metabolism
Culture Environment
bull There is growing evidence that the environment in which an embryo
develops can effect its metabolism epigenetic alterations and
developmental potential all aspects of environment important
bull Culture conditions have been shown in mouse models to impact
epigenetic patterns of the embryo and especially the placenta
bull Specifically culture at atmospheric (20) oxygen tension as compared to
physiologic (5) oxygen tension resulted in marked differences in global
gene expression in particular genes involved in cell growth and
maintenance relative to embryos developed in vivo
Donrsquot stress gametes or embryos Maintain homeostatic environment
Healthy adults from IVF should be
our end point
bull Embryos do have the ability to adapt to less than optimal culture
conditions however this comes at a cost culminating in incrementally
lower rates of blastocyst andor viability post implantation
bull Exposing preimplantation embryo to stressors that impair function of
mitochondria not only alters survival to the blastocyst and resultant
viability but subsequent fetal growth and health
Egg Retrieval
Once the follicular fluid has been
aspirated the eggs are removed
from the fluid in an IVF chamber
and placed into culture media
Eggs are retrieved trans-vaginally
with the use of a Ovum needle attached
to a vaginal ultrasound probe
Follicle Scan
eggs are within
the follicles
bull Number of follicles does not equal number
of eggs retrieved
bull Average egg number is around 8-10
bull Quality not quantity everyone is different
Egg Maturation
bull Only mature eggs will fertilise
Germinal Vesicle Metaphase I Metaphase II
Mature Egg
Maturation
Semen Parameters
SPERM CONCENTRATION
gt15 MILLION PER ML
Natural Variations
SPERM MOTILITY
gt50 Swimming forward
Different grades of motility
SPERM MORPHOLOGY
lt96 Abnormal forms considered Normal
Genetic
Lifestyle
ANTI-SPERM ANTIBODIES
Sperm Retrieval
bull Ejaculation ndash Most common - Motile sperm have to be separated from the non motile and abnormal
sperm using a two part gradient system and centrifugation
- Sperm are then washed with culture media and are ready
for insemination
If indicated
bull Retrograde Ejaculation
bull Sperm extraction
Males that may require intervention for sperm retrieval
Obstructive azoospermia
eg Vasectomy
Congenital Absence of Vas deference (CAV)
Non-obstructive azoospermia
eg Kleinfelters
Y-chromosome deletion
Sertoli cell only
Virtual Azoospermia
Depending on the aetiology sperm can be retrieved by
bull Percutaneous Epididymal Sperm Aspiration (PESA) (sperm stored in epididymus aspirated using needle)
bull Testicular Sperm Aspiration (TESA) (tubules in testis which produce sperm aspirated using needle)
bull Open Testicular Biopsy (incision made into scrotum exposing testis and tubules retrieved)
Can be performed under local anaesthetic unless
incision required then need general anaesthetic
Sperm Retrieval ndash Testicular biopsy
Seminiferous Tubules from Testicular Biopsy
Insemination
bull Depends on sperm quality or patient history (previous IVF cycles)
ndash Standard Insemination (IVF)
ndash Microinjection (ICSI)
Microinjection (ICSI)
In cases where semen quality is sub-optimal the
sperm are injected into the egg using a procedure
called Intra-Cytoplasmic Sperm Injection (ICSI)
A single sperm is immobilized and drawn into a fine pipette
for injection into the egg The egg is held steady using a
ldquoholdingrdquo pipette
The sperm is expelled into the cytoplasm of the egg and the
pipette is withdrawn from the egg
ICSI
Fertilisation
Fertilisation (Day 1)
Fertilised egg Unfertilised Abnormal
(2 Pronuclei Visible) (gt2 PN)
eg
- Standard Insemination more
than 1 sperm has entered
- ICSI ndash Second polar body
hasnrsquot
been extruded by egg as above
bull Pronuclei appear
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Differences in the physiology of the mammalian embryo for
development from the zygote to the blastocyst stage
Precompaction stage Postcompaction stage
Low biosynthetic activity High biosynthetic activity
Low QO2 (Metabolic Quotient) High QO2 (Metabolic Quotient)
Pyruvate-based metabolism Glucose-based metabolism
Maternal genome Embryonic genome
Single cell Transporting epithelium
Low ability to maintain cellular Complex systems for maintenance
homeostasis of cellular homeostasis
Totipotent Differentiation into inner cell mass
and trophectoderm
Oviduct Uterus
uarr pyruvate darrpyruvate
uarrlactate darr lactate
darrglucose uarr glucose
uarrnon-ess AA uarr non-ess AA
uarr essential AA
Does culture media matter if your patients are
getting pregnant
bull Evidence that there are differences in DNA methylation patterns in
placental and umbilical blood samples from IVF children
compared to naturally conceived
bull There is an association with ART and specific imprinting disorders
bull SubfertilityInfertility hormone stimulation in vitro culture uterine
environment appear to influence the proper establishment and
maintenance of genomic imprints in the developing epigenome
Epigenetics amp Imprinting
bull Epigenetics Chromatin modifications that regulate gene activity
that are not due to DNA sequence changes (Saitou et al 2012)
bull Imprinted genes are genes whose expression is determined by
the parent that contributed them
bull Imprinted genes violate the usual rule of inheritance that both
alleles in a heterozygote are equally expressed
bull Genomic imprinting is a specialized epigenetic mechanism that
employs repressive modifications to silence one parental allele
while activating modifications on the other parental allele enable
expression Hirasawa amp Feil 2010
bull DNA methylation and histone modifications are two epigenetic
mechanisms that alter the functional state of chromatin activating
or repressing gene expression
In Vitro Embryo culture ndash Culture Media
bull Does culture media effect the health of the embryo
bull Suboptimal media may compromising imprint maintenance
ndash Mouse embryos cultured in deficient media displayed
biallelicH19 expression in postimplantation extraembryonic tissues
(Sasaki et al 1995)
ndash Whittens but not potassium simplex optimized medium plus amino acids
(KSOMaa) cultured embryos exhibited biallelic H19 expression
(Doherty et al 2000)
bull Extremely important for culture media to provide all the nutritional
requirements when required
bull Metabolites and cofactors are important regulators of the
epigenome
bull Many imprinted genes are involved in growth and metabolism
Culture Environment
bull There is growing evidence that the environment in which an embryo
develops can effect its metabolism epigenetic alterations and
developmental potential all aspects of environment important
bull Culture conditions have been shown in mouse models to impact
epigenetic patterns of the embryo and especially the placenta
bull Specifically culture at atmospheric (20) oxygen tension as compared to
physiologic (5) oxygen tension resulted in marked differences in global
gene expression in particular genes involved in cell growth and
maintenance relative to embryos developed in vivo
Donrsquot stress gametes or embryos Maintain homeostatic environment
Healthy adults from IVF should be
our end point
bull Embryos do have the ability to adapt to less than optimal culture
conditions however this comes at a cost culminating in incrementally
lower rates of blastocyst andor viability post implantation
bull Exposing preimplantation embryo to stressors that impair function of
mitochondria not only alters survival to the blastocyst and resultant
viability but subsequent fetal growth and health
Egg Retrieval
Once the follicular fluid has been
aspirated the eggs are removed
from the fluid in an IVF chamber
and placed into culture media
Eggs are retrieved trans-vaginally
with the use of a Ovum needle attached
to a vaginal ultrasound probe
Follicle Scan
eggs are within
the follicles
bull Number of follicles does not equal number
of eggs retrieved
bull Average egg number is around 8-10
bull Quality not quantity everyone is different
Egg Maturation
bull Only mature eggs will fertilise
Germinal Vesicle Metaphase I Metaphase II
Mature Egg
Maturation
Semen Parameters
SPERM CONCENTRATION
gt15 MILLION PER ML
Natural Variations
SPERM MOTILITY
gt50 Swimming forward
Different grades of motility
SPERM MORPHOLOGY
lt96 Abnormal forms considered Normal
Genetic
Lifestyle
ANTI-SPERM ANTIBODIES
Sperm Retrieval
bull Ejaculation ndash Most common - Motile sperm have to be separated from the non motile and abnormal
sperm using a two part gradient system and centrifugation
- Sperm are then washed with culture media and are ready
for insemination
If indicated
bull Retrograde Ejaculation
bull Sperm extraction
Males that may require intervention for sperm retrieval
Obstructive azoospermia
eg Vasectomy
Congenital Absence of Vas deference (CAV)
Non-obstructive azoospermia
eg Kleinfelters
Y-chromosome deletion
Sertoli cell only
Virtual Azoospermia
Depending on the aetiology sperm can be retrieved by
bull Percutaneous Epididymal Sperm Aspiration (PESA) (sperm stored in epididymus aspirated using needle)
bull Testicular Sperm Aspiration (TESA) (tubules in testis which produce sperm aspirated using needle)
bull Open Testicular Biopsy (incision made into scrotum exposing testis and tubules retrieved)
Can be performed under local anaesthetic unless
incision required then need general anaesthetic
Sperm Retrieval ndash Testicular biopsy
Seminiferous Tubules from Testicular Biopsy
Insemination
bull Depends on sperm quality or patient history (previous IVF cycles)
ndash Standard Insemination (IVF)
ndash Microinjection (ICSI)
Microinjection (ICSI)
In cases where semen quality is sub-optimal the
sperm are injected into the egg using a procedure
called Intra-Cytoplasmic Sperm Injection (ICSI)
A single sperm is immobilized and drawn into a fine pipette
for injection into the egg The egg is held steady using a
ldquoholdingrdquo pipette
The sperm is expelled into the cytoplasm of the egg and the
pipette is withdrawn from the egg
ICSI
Fertilisation
Fertilisation (Day 1)
Fertilised egg Unfertilised Abnormal
(2 Pronuclei Visible) (gt2 PN)
eg
- Standard Insemination more
than 1 sperm has entered
- ICSI ndash Second polar body
hasnrsquot
been extruded by egg as above
bull Pronuclei appear
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Oviduct Uterus
uarr pyruvate darrpyruvate
uarrlactate darr lactate
darrglucose uarr glucose
uarrnon-ess AA uarr non-ess AA
uarr essential AA
Does culture media matter if your patients are
getting pregnant
bull Evidence that there are differences in DNA methylation patterns in
placental and umbilical blood samples from IVF children
compared to naturally conceived
bull There is an association with ART and specific imprinting disorders
bull SubfertilityInfertility hormone stimulation in vitro culture uterine
environment appear to influence the proper establishment and
maintenance of genomic imprints in the developing epigenome
Epigenetics amp Imprinting
bull Epigenetics Chromatin modifications that regulate gene activity
that are not due to DNA sequence changes (Saitou et al 2012)
bull Imprinted genes are genes whose expression is determined by
the parent that contributed them
bull Imprinted genes violate the usual rule of inheritance that both
alleles in a heterozygote are equally expressed
bull Genomic imprinting is a specialized epigenetic mechanism that
employs repressive modifications to silence one parental allele
while activating modifications on the other parental allele enable
expression Hirasawa amp Feil 2010
bull DNA methylation and histone modifications are two epigenetic
mechanisms that alter the functional state of chromatin activating
or repressing gene expression
In Vitro Embryo culture ndash Culture Media
bull Does culture media effect the health of the embryo
bull Suboptimal media may compromising imprint maintenance
ndash Mouse embryos cultured in deficient media displayed
biallelicH19 expression in postimplantation extraembryonic tissues
(Sasaki et al 1995)
ndash Whittens but not potassium simplex optimized medium plus amino acids
(KSOMaa) cultured embryos exhibited biallelic H19 expression
(Doherty et al 2000)
bull Extremely important for culture media to provide all the nutritional
requirements when required
bull Metabolites and cofactors are important regulators of the
epigenome
bull Many imprinted genes are involved in growth and metabolism
Culture Environment
bull There is growing evidence that the environment in which an embryo
develops can effect its metabolism epigenetic alterations and
developmental potential all aspects of environment important
bull Culture conditions have been shown in mouse models to impact
epigenetic patterns of the embryo and especially the placenta
bull Specifically culture at atmospheric (20) oxygen tension as compared to
physiologic (5) oxygen tension resulted in marked differences in global
gene expression in particular genes involved in cell growth and
maintenance relative to embryos developed in vivo
Donrsquot stress gametes or embryos Maintain homeostatic environment
Healthy adults from IVF should be
our end point
bull Embryos do have the ability to adapt to less than optimal culture
conditions however this comes at a cost culminating in incrementally
lower rates of blastocyst andor viability post implantation
bull Exposing preimplantation embryo to stressors that impair function of
mitochondria not only alters survival to the blastocyst and resultant
viability but subsequent fetal growth and health
Egg Retrieval
Once the follicular fluid has been
aspirated the eggs are removed
from the fluid in an IVF chamber
and placed into culture media
Eggs are retrieved trans-vaginally
with the use of a Ovum needle attached
to a vaginal ultrasound probe
Follicle Scan
eggs are within
the follicles
bull Number of follicles does not equal number
of eggs retrieved
bull Average egg number is around 8-10
bull Quality not quantity everyone is different
Egg Maturation
bull Only mature eggs will fertilise
Germinal Vesicle Metaphase I Metaphase II
Mature Egg
Maturation
Semen Parameters
SPERM CONCENTRATION
gt15 MILLION PER ML
Natural Variations
SPERM MOTILITY
gt50 Swimming forward
Different grades of motility
SPERM MORPHOLOGY
lt96 Abnormal forms considered Normal
Genetic
Lifestyle
ANTI-SPERM ANTIBODIES
Sperm Retrieval
bull Ejaculation ndash Most common - Motile sperm have to be separated from the non motile and abnormal
sperm using a two part gradient system and centrifugation
- Sperm are then washed with culture media and are ready
for insemination
If indicated
bull Retrograde Ejaculation
bull Sperm extraction
Males that may require intervention for sperm retrieval
Obstructive azoospermia
eg Vasectomy
Congenital Absence of Vas deference (CAV)
Non-obstructive azoospermia
eg Kleinfelters
Y-chromosome deletion
Sertoli cell only
Virtual Azoospermia
Depending on the aetiology sperm can be retrieved by
bull Percutaneous Epididymal Sperm Aspiration (PESA) (sperm stored in epididymus aspirated using needle)
bull Testicular Sperm Aspiration (TESA) (tubules in testis which produce sperm aspirated using needle)
bull Open Testicular Biopsy (incision made into scrotum exposing testis and tubules retrieved)
Can be performed under local anaesthetic unless
incision required then need general anaesthetic
Sperm Retrieval ndash Testicular biopsy
Seminiferous Tubules from Testicular Biopsy
Insemination
bull Depends on sperm quality or patient history (previous IVF cycles)
ndash Standard Insemination (IVF)
ndash Microinjection (ICSI)
Microinjection (ICSI)
In cases where semen quality is sub-optimal the
sperm are injected into the egg using a procedure
called Intra-Cytoplasmic Sperm Injection (ICSI)
A single sperm is immobilized and drawn into a fine pipette
for injection into the egg The egg is held steady using a
ldquoholdingrdquo pipette
The sperm is expelled into the cytoplasm of the egg and the
pipette is withdrawn from the egg
ICSI
Fertilisation
Fertilisation (Day 1)
Fertilised egg Unfertilised Abnormal
(2 Pronuclei Visible) (gt2 PN)
eg
- Standard Insemination more
than 1 sperm has entered
- ICSI ndash Second polar body
hasnrsquot
been extruded by egg as above
bull Pronuclei appear
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Does culture media matter if your patients are
getting pregnant
bull Evidence that there are differences in DNA methylation patterns in
placental and umbilical blood samples from IVF children
compared to naturally conceived
bull There is an association with ART and specific imprinting disorders
bull SubfertilityInfertility hormone stimulation in vitro culture uterine
environment appear to influence the proper establishment and
maintenance of genomic imprints in the developing epigenome
Epigenetics amp Imprinting
bull Epigenetics Chromatin modifications that regulate gene activity
that are not due to DNA sequence changes (Saitou et al 2012)
bull Imprinted genes are genes whose expression is determined by
the parent that contributed them
bull Imprinted genes violate the usual rule of inheritance that both
alleles in a heterozygote are equally expressed
bull Genomic imprinting is a specialized epigenetic mechanism that
employs repressive modifications to silence one parental allele
while activating modifications on the other parental allele enable
expression Hirasawa amp Feil 2010
bull DNA methylation and histone modifications are two epigenetic
mechanisms that alter the functional state of chromatin activating
or repressing gene expression
In Vitro Embryo culture ndash Culture Media
bull Does culture media effect the health of the embryo
bull Suboptimal media may compromising imprint maintenance
ndash Mouse embryos cultured in deficient media displayed
biallelicH19 expression in postimplantation extraembryonic tissues
(Sasaki et al 1995)
ndash Whittens but not potassium simplex optimized medium plus amino acids
(KSOMaa) cultured embryos exhibited biallelic H19 expression
(Doherty et al 2000)
bull Extremely important for culture media to provide all the nutritional
requirements when required
bull Metabolites and cofactors are important regulators of the
epigenome
bull Many imprinted genes are involved in growth and metabolism
Culture Environment
bull There is growing evidence that the environment in which an embryo
develops can effect its metabolism epigenetic alterations and
developmental potential all aspects of environment important
bull Culture conditions have been shown in mouse models to impact
epigenetic patterns of the embryo and especially the placenta
bull Specifically culture at atmospheric (20) oxygen tension as compared to
physiologic (5) oxygen tension resulted in marked differences in global
gene expression in particular genes involved in cell growth and
maintenance relative to embryos developed in vivo
Donrsquot stress gametes or embryos Maintain homeostatic environment
Healthy adults from IVF should be
our end point
bull Embryos do have the ability to adapt to less than optimal culture
conditions however this comes at a cost culminating in incrementally
lower rates of blastocyst andor viability post implantation
bull Exposing preimplantation embryo to stressors that impair function of
mitochondria not only alters survival to the blastocyst and resultant
viability but subsequent fetal growth and health
Egg Retrieval
Once the follicular fluid has been
aspirated the eggs are removed
from the fluid in an IVF chamber
and placed into culture media
Eggs are retrieved trans-vaginally
with the use of a Ovum needle attached
to a vaginal ultrasound probe
Follicle Scan
eggs are within
the follicles
bull Number of follicles does not equal number
of eggs retrieved
bull Average egg number is around 8-10
bull Quality not quantity everyone is different
Egg Maturation
bull Only mature eggs will fertilise
Germinal Vesicle Metaphase I Metaphase II
Mature Egg
Maturation
Semen Parameters
SPERM CONCENTRATION
gt15 MILLION PER ML
Natural Variations
SPERM MOTILITY
gt50 Swimming forward
Different grades of motility
SPERM MORPHOLOGY
lt96 Abnormal forms considered Normal
Genetic
Lifestyle
ANTI-SPERM ANTIBODIES
Sperm Retrieval
bull Ejaculation ndash Most common - Motile sperm have to be separated from the non motile and abnormal
sperm using a two part gradient system and centrifugation
- Sperm are then washed with culture media and are ready
for insemination
If indicated
bull Retrograde Ejaculation
bull Sperm extraction
Males that may require intervention for sperm retrieval
Obstructive azoospermia
eg Vasectomy
Congenital Absence of Vas deference (CAV)
Non-obstructive azoospermia
eg Kleinfelters
Y-chromosome deletion
Sertoli cell only
Virtual Azoospermia
Depending on the aetiology sperm can be retrieved by
bull Percutaneous Epididymal Sperm Aspiration (PESA) (sperm stored in epididymus aspirated using needle)
bull Testicular Sperm Aspiration (TESA) (tubules in testis which produce sperm aspirated using needle)
bull Open Testicular Biopsy (incision made into scrotum exposing testis and tubules retrieved)
Can be performed under local anaesthetic unless
incision required then need general anaesthetic
Sperm Retrieval ndash Testicular biopsy
Seminiferous Tubules from Testicular Biopsy
Insemination
bull Depends on sperm quality or patient history (previous IVF cycles)
ndash Standard Insemination (IVF)
ndash Microinjection (ICSI)
Microinjection (ICSI)
In cases where semen quality is sub-optimal the
sperm are injected into the egg using a procedure
called Intra-Cytoplasmic Sperm Injection (ICSI)
A single sperm is immobilized and drawn into a fine pipette
for injection into the egg The egg is held steady using a
ldquoholdingrdquo pipette
The sperm is expelled into the cytoplasm of the egg and the
pipette is withdrawn from the egg
ICSI
Fertilisation
Fertilisation (Day 1)
Fertilised egg Unfertilised Abnormal
(2 Pronuclei Visible) (gt2 PN)
eg
- Standard Insemination more
than 1 sperm has entered
- ICSI ndash Second polar body
hasnrsquot
been extruded by egg as above
bull Pronuclei appear
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Epigenetics amp Imprinting
bull Epigenetics Chromatin modifications that regulate gene activity
that are not due to DNA sequence changes (Saitou et al 2012)
bull Imprinted genes are genes whose expression is determined by
the parent that contributed them
bull Imprinted genes violate the usual rule of inheritance that both
alleles in a heterozygote are equally expressed
bull Genomic imprinting is a specialized epigenetic mechanism that
employs repressive modifications to silence one parental allele
while activating modifications on the other parental allele enable
expression Hirasawa amp Feil 2010
bull DNA methylation and histone modifications are two epigenetic
mechanisms that alter the functional state of chromatin activating
or repressing gene expression
In Vitro Embryo culture ndash Culture Media
bull Does culture media effect the health of the embryo
bull Suboptimal media may compromising imprint maintenance
ndash Mouse embryos cultured in deficient media displayed
biallelicH19 expression in postimplantation extraembryonic tissues
(Sasaki et al 1995)
ndash Whittens but not potassium simplex optimized medium plus amino acids
(KSOMaa) cultured embryos exhibited biallelic H19 expression
(Doherty et al 2000)
bull Extremely important for culture media to provide all the nutritional
requirements when required
bull Metabolites and cofactors are important regulators of the
epigenome
bull Many imprinted genes are involved in growth and metabolism
Culture Environment
bull There is growing evidence that the environment in which an embryo
develops can effect its metabolism epigenetic alterations and
developmental potential all aspects of environment important
bull Culture conditions have been shown in mouse models to impact
epigenetic patterns of the embryo and especially the placenta
bull Specifically culture at atmospheric (20) oxygen tension as compared to
physiologic (5) oxygen tension resulted in marked differences in global
gene expression in particular genes involved in cell growth and
maintenance relative to embryos developed in vivo
Donrsquot stress gametes or embryos Maintain homeostatic environment
Healthy adults from IVF should be
our end point
bull Embryos do have the ability to adapt to less than optimal culture
conditions however this comes at a cost culminating in incrementally
lower rates of blastocyst andor viability post implantation
bull Exposing preimplantation embryo to stressors that impair function of
mitochondria not only alters survival to the blastocyst and resultant
viability but subsequent fetal growth and health
Egg Retrieval
Once the follicular fluid has been
aspirated the eggs are removed
from the fluid in an IVF chamber
and placed into culture media
Eggs are retrieved trans-vaginally
with the use of a Ovum needle attached
to a vaginal ultrasound probe
Follicle Scan
eggs are within
the follicles
bull Number of follicles does not equal number
of eggs retrieved
bull Average egg number is around 8-10
bull Quality not quantity everyone is different
Egg Maturation
bull Only mature eggs will fertilise
Germinal Vesicle Metaphase I Metaphase II
Mature Egg
Maturation
Semen Parameters
SPERM CONCENTRATION
gt15 MILLION PER ML
Natural Variations
SPERM MOTILITY
gt50 Swimming forward
Different grades of motility
SPERM MORPHOLOGY
lt96 Abnormal forms considered Normal
Genetic
Lifestyle
ANTI-SPERM ANTIBODIES
Sperm Retrieval
bull Ejaculation ndash Most common - Motile sperm have to be separated from the non motile and abnormal
sperm using a two part gradient system and centrifugation
- Sperm are then washed with culture media and are ready
for insemination
If indicated
bull Retrograde Ejaculation
bull Sperm extraction
Males that may require intervention for sperm retrieval
Obstructive azoospermia
eg Vasectomy
Congenital Absence of Vas deference (CAV)
Non-obstructive azoospermia
eg Kleinfelters
Y-chromosome deletion
Sertoli cell only
Virtual Azoospermia
Depending on the aetiology sperm can be retrieved by
bull Percutaneous Epididymal Sperm Aspiration (PESA) (sperm stored in epididymus aspirated using needle)
bull Testicular Sperm Aspiration (TESA) (tubules in testis which produce sperm aspirated using needle)
bull Open Testicular Biopsy (incision made into scrotum exposing testis and tubules retrieved)
Can be performed under local anaesthetic unless
incision required then need general anaesthetic
Sperm Retrieval ndash Testicular biopsy
Seminiferous Tubules from Testicular Biopsy
Insemination
bull Depends on sperm quality or patient history (previous IVF cycles)
ndash Standard Insemination (IVF)
ndash Microinjection (ICSI)
Microinjection (ICSI)
In cases where semen quality is sub-optimal the
sperm are injected into the egg using a procedure
called Intra-Cytoplasmic Sperm Injection (ICSI)
A single sperm is immobilized and drawn into a fine pipette
for injection into the egg The egg is held steady using a
ldquoholdingrdquo pipette
The sperm is expelled into the cytoplasm of the egg and the
pipette is withdrawn from the egg
ICSI
Fertilisation
Fertilisation (Day 1)
Fertilised egg Unfertilised Abnormal
(2 Pronuclei Visible) (gt2 PN)
eg
- Standard Insemination more
than 1 sperm has entered
- ICSI ndash Second polar body
hasnrsquot
been extruded by egg as above
bull Pronuclei appear
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
In Vitro Embryo culture ndash Culture Media
bull Does culture media effect the health of the embryo
bull Suboptimal media may compromising imprint maintenance
ndash Mouse embryos cultured in deficient media displayed
biallelicH19 expression in postimplantation extraembryonic tissues
(Sasaki et al 1995)
ndash Whittens but not potassium simplex optimized medium plus amino acids
(KSOMaa) cultured embryos exhibited biallelic H19 expression
(Doherty et al 2000)
bull Extremely important for culture media to provide all the nutritional
requirements when required
bull Metabolites and cofactors are important regulators of the
epigenome
bull Many imprinted genes are involved in growth and metabolism
Culture Environment
bull There is growing evidence that the environment in which an embryo
develops can effect its metabolism epigenetic alterations and
developmental potential all aspects of environment important
bull Culture conditions have been shown in mouse models to impact
epigenetic patterns of the embryo and especially the placenta
bull Specifically culture at atmospheric (20) oxygen tension as compared to
physiologic (5) oxygen tension resulted in marked differences in global
gene expression in particular genes involved in cell growth and
maintenance relative to embryos developed in vivo
Donrsquot stress gametes or embryos Maintain homeostatic environment
Healthy adults from IVF should be
our end point
bull Embryos do have the ability to adapt to less than optimal culture
conditions however this comes at a cost culminating in incrementally
lower rates of blastocyst andor viability post implantation
bull Exposing preimplantation embryo to stressors that impair function of
mitochondria not only alters survival to the blastocyst and resultant
viability but subsequent fetal growth and health
Egg Retrieval
Once the follicular fluid has been
aspirated the eggs are removed
from the fluid in an IVF chamber
and placed into culture media
Eggs are retrieved trans-vaginally
with the use of a Ovum needle attached
to a vaginal ultrasound probe
Follicle Scan
eggs are within
the follicles
bull Number of follicles does not equal number
of eggs retrieved
bull Average egg number is around 8-10
bull Quality not quantity everyone is different
Egg Maturation
bull Only mature eggs will fertilise
Germinal Vesicle Metaphase I Metaphase II
Mature Egg
Maturation
Semen Parameters
SPERM CONCENTRATION
gt15 MILLION PER ML
Natural Variations
SPERM MOTILITY
gt50 Swimming forward
Different grades of motility
SPERM MORPHOLOGY
lt96 Abnormal forms considered Normal
Genetic
Lifestyle
ANTI-SPERM ANTIBODIES
Sperm Retrieval
bull Ejaculation ndash Most common - Motile sperm have to be separated from the non motile and abnormal
sperm using a two part gradient system and centrifugation
- Sperm are then washed with culture media and are ready
for insemination
If indicated
bull Retrograde Ejaculation
bull Sperm extraction
Males that may require intervention for sperm retrieval
Obstructive azoospermia
eg Vasectomy
Congenital Absence of Vas deference (CAV)
Non-obstructive azoospermia
eg Kleinfelters
Y-chromosome deletion
Sertoli cell only
Virtual Azoospermia
Depending on the aetiology sperm can be retrieved by
bull Percutaneous Epididymal Sperm Aspiration (PESA) (sperm stored in epididymus aspirated using needle)
bull Testicular Sperm Aspiration (TESA) (tubules in testis which produce sperm aspirated using needle)
bull Open Testicular Biopsy (incision made into scrotum exposing testis and tubules retrieved)
Can be performed under local anaesthetic unless
incision required then need general anaesthetic
Sperm Retrieval ndash Testicular biopsy
Seminiferous Tubules from Testicular Biopsy
Insemination
bull Depends on sperm quality or patient history (previous IVF cycles)
ndash Standard Insemination (IVF)
ndash Microinjection (ICSI)
Microinjection (ICSI)
In cases where semen quality is sub-optimal the
sperm are injected into the egg using a procedure
called Intra-Cytoplasmic Sperm Injection (ICSI)
A single sperm is immobilized and drawn into a fine pipette
for injection into the egg The egg is held steady using a
ldquoholdingrdquo pipette
The sperm is expelled into the cytoplasm of the egg and the
pipette is withdrawn from the egg
ICSI
Fertilisation
Fertilisation (Day 1)
Fertilised egg Unfertilised Abnormal
(2 Pronuclei Visible) (gt2 PN)
eg
- Standard Insemination more
than 1 sperm has entered
- ICSI ndash Second polar body
hasnrsquot
been extruded by egg as above
bull Pronuclei appear
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Culture Environment
bull There is growing evidence that the environment in which an embryo
develops can effect its metabolism epigenetic alterations and
developmental potential all aspects of environment important
bull Culture conditions have been shown in mouse models to impact
epigenetic patterns of the embryo and especially the placenta
bull Specifically culture at atmospheric (20) oxygen tension as compared to
physiologic (5) oxygen tension resulted in marked differences in global
gene expression in particular genes involved in cell growth and
maintenance relative to embryos developed in vivo
Donrsquot stress gametes or embryos Maintain homeostatic environment
Healthy adults from IVF should be
our end point
bull Embryos do have the ability to adapt to less than optimal culture
conditions however this comes at a cost culminating in incrementally
lower rates of blastocyst andor viability post implantation
bull Exposing preimplantation embryo to stressors that impair function of
mitochondria not only alters survival to the blastocyst and resultant
viability but subsequent fetal growth and health
Egg Retrieval
Once the follicular fluid has been
aspirated the eggs are removed
from the fluid in an IVF chamber
and placed into culture media
Eggs are retrieved trans-vaginally
with the use of a Ovum needle attached
to a vaginal ultrasound probe
Follicle Scan
eggs are within
the follicles
bull Number of follicles does not equal number
of eggs retrieved
bull Average egg number is around 8-10
bull Quality not quantity everyone is different
Egg Maturation
bull Only mature eggs will fertilise
Germinal Vesicle Metaphase I Metaphase II
Mature Egg
Maturation
Semen Parameters
SPERM CONCENTRATION
gt15 MILLION PER ML
Natural Variations
SPERM MOTILITY
gt50 Swimming forward
Different grades of motility
SPERM MORPHOLOGY
lt96 Abnormal forms considered Normal
Genetic
Lifestyle
ANTI-SPERM ANTIBODIES
Sperm Retrieval
bull Ejaculation ndash Most common - Motile sperm have to be separated from the non motile and abnormal
sperm using a two part gradient system and centrifugation
- Sperm are then washed with culture media and are ready
for insemination
If indicated
bull Retrograde Ejaculation
bull Sperm extraction
Males that may require intervention for sperm retrieval
Obstructive azoospermia
eg Vasectomy
Congenital Absence of Vas deference (CAV)
Non-obstructive azoospermia
eg Kleinfelters
Y-chromosome deletion
Sertoli cell only
Virtual Azoospermia
Depending on the aetiology sperm can be retrieved by
bull Percutaneous Epididymal Sperm Aspiration (PESA) (sperm stored in epididymus aspirated using needle)
bull Testicular Sperm Aspiration (TESA) (tubules in testis which produce sperm aspirated using needle)
bull Open Testicular Biopsy (incision made into scrotum exposing testis and tubules retrieved)
Can be performed under local anaesthetic unless
incision required then need general anaesthetic
Sperm Retrieval ndash Testicular biopsy
Seminiferous Tubules from Testicular Biopsy
Insemination
bull Depends on sperm quality or patient history (previous IVF cycles)
ndash Standard Insemination (IVF)
ndash Microinjection (ICSI)
Microinjection (ICSI)
In cases where semen quality is sub-optimal the
sperm are injected into the egg using a procedure
called Intra-Cytoplasmic Sperm Injection (ICSI)
A single sperm is immobilized and drawn into a fine pipette
for injection into the egg The egg is held steady using a
ldquoholdingrdquo pipette
The sperm is expelled into the cytoplasm of the egg and the
pipette is withdrawn from the egg
ICSI
Fertilisation
Fertilisation (Day 1)
Fertilised egg Unfertilised Abnormal
(2 Pronuclei Visible) (gt2 PN)
eg
- Standard Insemination more
than 1 sperm has entered
- ICSI ndash Second polar body
hasnrsquot
been extruded by egg as above
bull Pronuclei appear
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Healthy adults from IVF should be
our end point
bull Embryos do have the ability to adapt to less than optimal culture
conditions however this comes at a cost culminating in incrementally
lower rates of blastocyst andor viability post implantation
bull Exposing preimplantation embryo to stressors that impair function of
mitochondria not only alters survival to the blastocyst and resultant
viability but subsequent fetal growth and health
Egg Retrieval
Once the follicular fluid has been
aspirated the eggs are removed
from the fluid in an IVF chamber
and placed into culture media
Eggs are retrieved trans-vaginally
with the use of a Ovum needle attached
to a vaginal ultrasound probe
Follicle Scan
eggs are within
the follicles
bull Number of follicles does not equal number
of eggs retrieved
bull Average egg number is around 8-10
bull Quality not quantity everyone is different
Egg Maturation
bull Only mature eggs will fertilise
Germinal Vesicle Metaphase I Metaphase II
Mature Egg
Maturation
Semen Parameters
SPERM CONCENTRATION
gt15 MILLION PER ML
Natural Variations
SPERM MOTILITY
gt50 Swimming forward
Different grades of motility
SPERM MORPHOLOGY
lt96 Abnormal forms considered Normal
Genetic
Lifestyle
ANTI-SPERM ANTIBODIES
Sperm Retrieval
bull Ejaculation ndash Most common - Motile sperm have to be separated from the non motile and abnormal
sperm using a two part gradient system and centrifugation
- Sperm are then washed with culture media and are ready
for insemination
If indicated
bull Retrograde Ejaculation
bull Sperm extraction
Males that may require intervention for sperm retrieval
Obstructive azoospermia
eg Vasectomy
Congenital Absence of Vas deference (CAV)
Non-obstructive azoospermia
eg Kleinfelters
Y-chromosome deletion
Sertoli cell only
Virtual Azoospermia
Depending on the aetiology sperm can be retrieved by
bull Percutaneous Epididymal Sperm Aspiration (PESA) (sperm stored in epididymus aspirated using needle)
bull Testicular Sperm Aspiration (TESA) (tubules in testis which produce sperm aspirated using needle)
bull Open Testicular Biopsy (incision made into scrotum exposing testis and tubules retrieved)
Can be performed under local anaesthetic unless
incision required then need general anaesthetic
Sperm Retrieval ndash Testicular biopsy
Seminiferous Tubules from Testicular Biopsy
Insemination
bull Depends on sperm quality or patient history (previous IVF cycles)
ndash Standard Insemination (IVF)
ndash Microinjection (ICSI)
Microinjection (ICSI)
In cases where semen quality is sub-optimal the
sperm are injected into the egg using a procedure
called Intra-Cytoplasmic Sperm Injection (ICSI)
A single sperm is immobilized and drawn into a fine pipette
for injection into the egg The egg is held steady using a
ldquoholdingrdquo pipette
The sperm is expelled into the cytoplasm of the egg and the
pipette is withdrawn from the egg
ICSI
Fertilisation
Fertilisation (Day 1)
Fertilised egg Unfertilised Abnormal
(2 Pronuclei Visible) (gt2 PN)
eg
- Standard Insemination more
than 1 sperm has entered
- ICSI ndash Second polar body
hasnrsquot
been extruded by egg as above
bull Pronuclei appear
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Egg Retrieval
Once the follicular fluid has been
aspirated the eggs are removed
from the fluid in an IVF chamber
and placed into culture media
Eggs are retrieved trans-vaginally
with the use of a Ovum needle attached
to a vaginal ultrasound probe
Follicle Scan
eggs are within
the follicles
bull Number of follicles does not equal number
of eggs retrieved
bull Average egg number is around 8-10
bull Quality not quantity everyone is different
Egg Maturation
bull Only mature eggs will fertilise
Germinal Vesicle Metaphase I Metaphase II
Mature Egg
Maturation
Semen Parameters
SPERM CONCENTRATION
gt15 MILLION PER ML
Natural Variations
SPERM MOTILITY
gt50 Swimming forward
Different grades of motility
SPERM MORPHOLOGY
lt96 Abnormal forms considered Normal
Genetic
Lifestyle
ANTI-SPERM ANTIBODIES
Sperm Retrieval
bull Ejaculation ndash Most common - Motile sperm have to be separated from the non motile and abnormal
sperm using a two part gradient system and centrifugation
- Sperm are then washed with culture media and are ready
for insemination
If indicated
bull Retrograde Ejaculation
bull Sperm extraction
Males that may require intervention for sperm retrieval
Obstructive azoospermia
eg Vasectomy
Congenital Absence of Vas deference (CAV)
Non-obstructive azoospermia
eg Kleinfelters
Y-chromosome deletion
Sertoli cell only
Virtual Azoospermia
Depending on the aetiology sperm can be retrieved by
bull Percutaneous Epididymal Sperm Aspiration (PESA) (sperm stored in epididymus aspirated using needle)
bull Testicular Sperm Aspiration (TESA) (tubules in testis which produce sperm aspirated using needle)
bull Open Testicular Biopsy (incision made into scrotum exposing testis and tubules retrieved)
Can be performed under local anaesthetic unless
incision required then need general anaesthetic
Sperm Retrieval ndash Testicular biopsy
Seminiferous Tubules from Testicular Biopsy
Insemination
bull Depends on sperm quality or patient history (previous IVF cycles)
ndash Standard Insemination (IVF)
ndash Microinjection (ICSI)
Microinjection (ICSI)
In cases where semen quality is sub-optimal the
sperm are injected into the egg using a procedure
called Intra-Cytoplasmic Sperm Injection (ICSI)
A single sperm is immobilized and drawn into a fine pipette
for injection into the egg The egg is held steady using a
ldquoholdingrdquo pipette
The sperm is expelled into the cytoplasm of the egg and the
pipette is withdrawn from the egg
ICSI
Fertilisation
Fertilisation (Day 1)
Fertilised egg Unfertilised Abnormal
(2 Pronuclei Visible) (gt2 PN)
eg
- Standard Insemination more
than 1 sperm has entered
- ICSI ndash Second polar body
hasnrsquot
been extruded by egg as above
bull Pronuclei appear
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Egg Maturation
bull Only mature eggs will fertilise
Germinal Vesicle Metaphase I Metaphase II
Mature Egg
Maturation
Semen Parameters
SPERM CONCENTRATION
gt15 MILLION PER ML
Natural Variations
SPERM MOTILITY
gt50 Swimming forward
Different grades of motility
SPERM MORPHOLOGY
lt96 Abnormal forms considered Normal
Genetic
Lifestyle
ANTI-SPERM ANTIBODIES
Sperm Retrieval
bull Ejaculation ndash Most common - Motile sperm have to be separated from the non motile and abnormal
sperm using a two part gradient system and centrifugation
- Sperm are then washed with culture media and are ready
for insemination
If indicated
bull Retrograde Ejaculation
bull Sperm extraction
Males that may require intervention for sperm retrieval
Obstructive azoospermia
eg Vasectomy
Congenital Absence of Vas deference (CAV)
Non-obstructive azoospermia
eg Kleinfelters
Y-chromosome deletion
Sertoli cell only
Virtual Azoospermia
Depending on the aetiology sperm can be retrieved by
bull Percutaneous Epididymal Sperm Aspiration (PESA) (sperm stored in epididymus aspirated using needle)
bull Testicular Sperm Aspiration (TESA) (tubules in testis which produce sperm aspirated using needle)
bull Open Testicular Biopsy (incision made into scrotum exposing testis and tubules retrieved)
Can be performed under local anaesthetic unless
incision required then need general anaesthetic
Sperm Retrieval ndash Testicular biopsy
Seminiferous Tubules from Testicular Biopsy
Insemination
bull Depends on sperm quality or patient history (previous IVF cycles)
ndash Standard Insemination (IVF)
ndash Microinjection (ICSI)
Microinjection (ICSI)
In cases where semen quality is sub-optimal the
sperm are injected into the egg using a procedure
called Intra-Cytoplasmic Sperm Injection (ICSI)
A single sperm is immobilized and drawn into a fine pipette
for injection into the egg The egg is held steady using a
ldquoholdingrdquo pipette
The sperm is expelled into the cytoplasm of the egg and the
pipette is withdrawn from the egg
ICSI
Fertilisation
Fertilisation (Day 1)
Fertilised egg Unfertilised Abnormal
(2 Pronuclei Visible) (gt2 PN)
eg
- Standard Insemination more
than 1 sperm has entered
- ICSI ndash Second polar body
hasnrsquot
been extruded by egg as above
bull Pronuclei appear
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Maturation
Semen Parameters
SPERM CONCENTRATION
gt15 MILLION PER ML
Natural Variations
SPERM MOTILITY
gt50 Swimming forward
Different grades of motility
SPERM MORPHOLOGY
lt96 Abnormal forms considered Normal
Genetic
Lifestyle
ANTI-SPERM ANTIBODIES
Sperm Retrieval
bull Ejaculation ndash Most common - Motile sperm have to be separated from the non motile and abnormal
sperm using a two part gradient system and centrifugation
- Sperm are then washed with culture media and are ready
for insemination
If indicated
bull Retrograde Ejaculation
bull Sperm extraction
Males that may require intervention for sperm retrieval
Obstructive azoospermia
eg Vasectomy
Congenital Absence of Vas deference (CAV)
Non-obstructive azoospermia
eg Kleinfelters
Y-chromosome deletion
Sertoli cell only
Virtual Azoospermia
Depending on the aetiology sperm can be retrieved by
bull Percutaneous Epididymal Sperm Aspiration (PESA) (sperm stored in epididymus aspirated using needle)
bull Testicular Sperm Aspiration (TESA) (tubules in testis which produce sperm aspirated using needle)
bull Open Testicular Biopsy (incision made into scrotum exposing testis and tubules retrieved)
Can be performed under local anaesthetic unless
incision required then need general anaesthetic
Sperm Retrieval ndash Testicular biopsy
Seminiferous Tubules from Testicular Biopsy
Insemination
bull Depends on sperm quality or patient history (previous IVF cycles)
ndash Standard Insemination (IVF)
ndash Microinjection (ICSI)
Microinjection (ICSI)
In cases where semen quality is sub-optimal the
sperm are injected into the egg using a procedure
called Intra-Cytoplasmic Sperm Injection (ICSI)
A single sperm is immobilized and drawn into a fine pipette
for injection into the egg The egg is held steady using a
ldquoholdingrdquo pipette
The sperm is expelled into the cytoplasm of the egg and the
pipette is withdrawn from the egg
ICSI
Fertilisation
Fertilisation (Day 1)
Fertilised egg Unfertilised Abnormal
(2 Pronuclei Visible) (gt2 PN)
eg
- Standard Insemination more
than 1 sperm has entered
- ICSI ndash Second polar body
hasnrsquot
been extruded by egg as above
bull Pronuclei appear
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Semen Parameters
SPERM CONCENTRATION
gt15 MILLION PER ML
Natural Variations
SPERM MOTILITY
gt50 Swimming forward
Different grades of motility
SPERM MORPHOLOGY
lt96 Abnormal forms considered Normal
Genetic
Lifestyle
ANTI-SPERM ANTIBODIES
Sperm Retrieval
bull Ejaculation ndash Most common - Motile sperm have to be separated from the non motile and abnormal
sperm using a two part gradient system and centrifugation
- Sperm are then washed with culture media and are ready
for insemination
If indicated
bull Retrograde Ejaculation
bull Sperm extraction
Males that may require intervention for sperm retrieval
Obstructive azoospermia
eg Vasectomy
Congenital Absence of Vas deference (CAV)
Non-obstructive azoospermia
eg Kleinfelters
Y-chromosome deletion
Sertoli cell only
Virtual Azoospermia
Depending on the aetiology sperm can be retrieved by
bull Percutaneous Epididymal Sperm Aspiration (PESA) (sperm stored in epididymus aspirated using needle)
bull Testicular Sperm Aspiration (TESA) (tubules in testis which produce sperm aspirated using needle)
bull Open Testicular Biopsy (incision made into scrotum exposing testis and tubules retrieved)
Can be performed under local anaesthetic unless
incision required then need general anaesthetic
Sperm Retrieval ndash Testicular biopsy
Seminiferous Tubules from Testicular Biopsy
Insemination
bull Depends on sperm quality or patient history (previous IVF cycles)
ndash Standard Insemination (IVF)
ndash Microinjection (ICSI)
Microinjection (ICSI)
In cases where semen quality is sub-optimal the
sperm are injected into the egg using a procedure
called Intra-Cytoplasmic Sperm Injection (ICSI)
A single sperm is immobilized and drawn into a fine pipette
for injection into the egg The egg is held steady using a
ldquoholdingrdquo pipette
The sperm is expelled into the cytoplasm of the egg and the
pipette is withdrawn from the egg
ICSI
Fertilisation
Fertilisation (Day 1)
Fertilised egg Unfertilised Abnormal
(2 Pronuclei Visible) (gt2 PN)
eg
- Standard Insemination more
than 1 sperm has entered
- ICSI ndash Second polar body
hasnrsquot
been extruded by egg as above
bull Pronuclei appear
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Sperm Retrieval
bull Ejaculation ndash Most common - Motile sperm have to be separated from the non motile and abnormal
sperm using a two part gradient system and centrifugation
- Sperm are then washed with culture media and are ready
for insemination
If indicated
bull Retrograde Ejaculation
bull Sperm extraction
Males that may require intervention for sperm retrieval
Obstructive azoospermia
eg Vasectomy
Congenital Absence of Vas deference (CAV)
Non-obstructive azoospermia
eg Kleinfelters
Y-chromosome deletion
Sertoli cell only
Virtual Azoospermia
Depending on the aetiology sperm can be retrieved by
bull Percutaneous Epididymal Sperm Aspiration (PESA) (sperm stored in epididymus aspirated using needle)
bull Testicular Sperm Aspiration (TESA) (tubules in testis which produce sperm aspirated using needle)
bull Open Testicular Biopsy (incision made into scrotum exposing testis and tubules retrieved)
Can be performed under local anaesthetic unless
incision required then need general anaesthetic
Sperm Retrieval ndash Testicular biopsy
Seminiferous Tubules from Testicular Biopsy
Insemination
bull Depends on sperm quality or patient history (previous IVF cycles)
ndash Standard Insemination (IVF)
ndash Microinjection (ICSI)
Microinjection (ICSI)
In cases where semen quality is sub-optimal the
sperm are injected into the egg using a procedure
called Intra-Cytoplasmic Sperm Injection (ICSI)
A single sperm is immobilized and drawn into a fine pipette
for injection into the egg The egg is held steady using a
ldquoholdingrdquo pipette
The sperm is expelled into the cytoplasm of the egg and the
pipette is withdrawn from the egg
ICSI
Fertilisation
Fertilisation (Day 1)
Fertilised egg Unfertilised Abnormal
(2 Pronuclei Visible) (gt2 PN)
eg
- Standard Insemination more
than 1 sperm has entered
- ICSI ndash Second polar body
hasnrsquot
been extruded by egg as above
bull Pronuclei appear
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Males that may require intervention for sperm retrieval
Obstructive azoospermia
eg Vasectomy
Congenital Absence of Vas deference (CAV)
Non-obstructive azoospermia
eg Kleinfelters
Y-chromosome deletion
Sertoli cell only
Virtual Azoospermia
Depending on the aetiology sperm can be retrieved by
bull Percutaneous Epididymal Sperm Aspiration (PESA) (sperm stored in epididymus aspirated using needle)
bull Testicular Sperm Aspiration (TESA) (tubules in testis which produce sperm aspirated using needle)
bull Open Testicular Biopsy (incision made into scrotum exposing testis and tubules retrieved)
Can be performed under local anaesthetic unless
incision required then need general anaesthetic
Sperm Retrieval ndash Testicular biopsy
Seminiferous Tubules from Testicular Biopsy
Insemination
bull Depends on sperm quality or patient history (previous IVF cycles)
ndash Standard Insemination (IVF)
ndash Microinjection (ICSI)
Microinjection (ICSI)
In cases where semen quality is sub-optimal the
sperm are injected into the egg using a procedure
called Intra-Cytoplasmic Sperm Injection (ICSI)
A single sperm is immobilized and drawn into a fine pipette
for injection into the egg The egg is held steady using a
ldquoholdingrdquo pipette
The sperm is expelled into the cytoplasm of the egg and the
pipette is withdrawn from the egg
ICSI
Fertilisation
Fertilisation (Day 1)
Fertilised egg Unfertilised Abnormal
(2 Pronuclei Visible) (gt2 PN)
eg
- Standard Insemination more
than 1 sperm has entered
- ICSI ndash Second polar body
hasnrsquot
been extruded by egg as above
bull Pronuclei appear
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Seminiferous Tubules from Testicular Biopsy
Insemination
bull Depends on sperm quality or patient history (previous IVF cycles)
ndash Standard Insemination (IVF)
ndash Microinjection (ICSI)
Microinjection (ICSI)
In cases where semen quality is sub-optimal the
sperm are injected into the egg using a procedure
called Intra-Cytoplasmic Sperm Injection (ICSI)
A single sperm is immobilized and drawn into a fine pipette
for injection into the egg The egg is held steady using a
ldquoholdingrdquo pipette
The sperm is expelled into the cytoplasm of the egg and the
pipette is withdrawn from the egg
ICSI
Fertilisation
Fertilisation (Day 1)
Fertilised egg Unfertilised Abnormal
(2 Pronuclei Visible) (gt2 PN)
eg
- Standard Insemination more
than 1 sperm has entered
- ICSI ndash Second polar body
hasnrsquot
been extruded by egg as above
bull Pronuclei appear
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Insemination
bull Depends on sperm quality or patient history (previous IVF cycles)
ndash Standard Insemination (IVF)
ndash Microinjection (ICSI)
Microinjection (ICSI)
In cases where semen quality is sub-optimal the
sperm are injected into the egg using a procedure
called Intra-Cytoplasmic Sperm Injection (ICSI)
A single sperm is immobilized and drawn into a fine pipette
for injection into the egg The egg is held steady using a
ldquoholdingrdquo pipette
The sperm is expelled into the cytoplasm of the egg and the
pipette is withdrawn from the egg
ICSI
Fertilisation
Fertilisation (Day 1)
Fertilised egg Unfertilised Abnormal
(2 Pronuclei Visible) (gt2 PN)
eg
- Standard Insemination more
than 1 sperm has entered
- ICSI ndash Second polar body
hasnrsquot
been extruded by egg as above
bull Pronuclei appear
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Microinjection (ICSI)
In cases where semen quality is sub-optimal the
sperm are injected into the egg using a procedure
called Intra-Cytoplasmic Sperm Injection (ICSI)
A single sperm is immobilized and drawn into a fine pipette
for injection into the egg The egg is held steady using a
ldquoholdingrdquo pipette
The sperm is expelled into the cytoplasm of the egg and the
pipette is withdrawn from the egg
ICSI
Fertilisation
Fertilisation (Day 1)
Fertilised egg Unfertilised Abnormal
(2 Pronuclei Visible) (gt2 PN)
eg
- Standard Insemination more
than 1 sperm has entered
- ICSI ndash Second polar body
hasnrsquot
been extruded by egg as above
bull Pronuclei appear
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
ICSI
Fertilisation
Fertilisation (Day 1)
Fertilised egg Unfertilised Abnormal
(2 Pronuclei Visible) (gt2 PN)
eg
- Standard Insemination more
than 1 sperm has entered
- ICSI ndash Second polar body
hasnrsquot
been extruded by egg as above
bull Pronuclei appear
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Fertilisation
Fertilisation (Day 1)
Fertilised egg Unfertilised Abnormal
(2 Pronuclei Visible) (gt2 PN)
eg
- Standard Insemination more
than 1 sperm has entered
- ICSI ndash Second polar body
hasnrsquot
been extruded by egg as above
bull Pronuclei appear
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Fertilisation (Day 1)
Fertilised egg Unfertilised Abnormal
(2 Pronuclei Visible) (gt2 PN)
eg
- Standard Insemination more
than 1 sperm has entered
- ICSI ndash Second polar body
hasnrsquot
been extruded by egg as above
bull Pronuclei appear
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
bull Pronuclei appear
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Sometimes things donrsquot go quite to planhellip
bull One pronuclei
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Failed Fertilisation
bull Can be due to the sperm egg or both
bull Standard Insemination Sperm egg interaction fails due to adhesion
binding penetration of zona pellucida or fusion with oolemma
ndash Lock and key defective eg egg may not have ZP3 receptor so sperm
cannot bind
ndash ~64 of males with abnormal sperm parameters have defective
sperm-zona interactions
ndash Most can be overcome with ICSI
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Failed Fertilisation with ICSI
bull ICSI Sperm able to activate egg or egg receptive to activation
Cascade of reactions required for fertilisation and pronuclear formation
ndash Defective sperm decondensation
ndash Eggs may have abnormal chromosome complements that donrsquot allow
further development
ndash Spindle which allows the chromosomes to split appropriately may be
abnormal and be a cause of failed fertilisation
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Abnormal Fertilisation
bull 3+ pronuclei
bull Standard IVF insemination 2+ sperm fertilising egg
ndash Failure of egg polyspermy mechanism egg has
several mechanisms to prevent more than one sperm
entering however in some instance they may fail
bull ICSI
ndash Failure of second polar body extrusion
bull Polyploidy mainly triploidy usually due to polyspermy occurs in about 2-3
of all human pregnancies and ~15 of miscarriages
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Day 2
bull Embryo starts dividing 24 hours post sperm insemination
bull Division during pre-implantation development is called cleavage
bull Day 2 embryo are expected to be at the 2-4 cell stage
Embryo Development
2-cell embryo 4-cell embryo
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Day 3
bull Embryo expected to be at the 6-8 cell stage
bull Switch from maternal to embryonic control
bull Quality of embryo dependent on
ndash Cell Stage
ndash Development
ndash Fragmentation
8-cell embryo
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Embryo Fragmentation
- During cell division fragments of cytoplasm break off
Programmed cell death
Cytoskeletal and spindle defects
- Extensive fragmentation have decreased blastulation rate gt15
fragmentation blastocyst rate declines
- Correlation with fragmentation and embryos that have inherent defects
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Day 4
bull Embryo at the Morula stage (mulberry like in appearance)
bull Embryo undergoes rapid cleavage
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Day 5 ndash Blastocyst
bull Not all Embryo can develop to the blastocyst stage
Therefore is used as a selection tool of most viable embryo
bull Embryo differentiates into 2 different cell types
bull Inner Cell Mass (ICM)
bull Trophectoderm (TE)
bull gt60 cells
bull Quality determined by
bull Approx no cells within the 2 cell types
bull Degeneration present
bull Expansion
ICM
TE
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Blastocysts come in different shapes and sizes
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Embryo Development
Day 1 Day 2
Day 3 Day 4 Day 5
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
bull Ultrasound guided
bull Embryo is aspirated into tip of catheter
bull Embryo injected into uterus
Embryo Transfer
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Cryopreservation
bull There are two methods of embryo cryopreservation
ndash Slow Freezing
ndash Vitrification
Nature 305 707 - 709 (1983) Human pregnancy following cryopreservation thawing and transfer of an eight-cell embryo Alan Trounson amp Linda Mohr Cryobiology 1984 Aug21(4)407-26 Vitrification as an approach to cryopreservationFahy GM MacFarlane DR Angell CA Meryman HT
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Slow Freezing
bull Was main method of cryopreservation in IVF for many years
bull Still used in some clinics with good results
bull Controlled step wise decrease in temperature
bull Lethal intracellular freezing can be avoided if cooling is slow
enough to permit sufficient water to leave the cell during
progressive freezing of the extracellular fluid
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
bull A process of converting a material into a glass-like amorphous solid
which is free of any crystalline structure either by the quick removal or
addition of heat or by mixing with an additive
bull Embryos suitable for freezing are vitrified to be used during a Frozen
Embryo Transfer cycle
bull Vitrification gives implantation rates of gt35 for blastocysts that have
been frozen stored at -196oC and warmed
bull Survival Rates in excess of 90 for oocytes cleavage embryos and
blastocysts
Vitrification
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
bull The major players in penetrating cryoprotectants
bull Ethylene Glycol
bull Dimethyl sulfoxide
bull 12-Propanediol
bull Time vs temperature vs concentration
bull Single vs cocktail
Cryoprotectants
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
bull Chinese Hamster Ovary cell line
bull Results showed
bull DMSO was not genotoxic
bull EG not directly genotoxic
bull PrOH produced DNA damage leading to
chromosome mutations
Cryoprotectants
Food Chem Toxicol 2010 Jul48(7)1905-12 doi 101016jfct201004032 Epub 2010 Apr 28
Assessment of the genotoxicity of three cryoprotectants used for
human oocyte vitrification dimethyl sulfoxide ethylene glycol and
propylene glycol Aye M1 Di Giorgio C De Mo M Botta A Perrin J Courbiere B
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Mol Hum Reprod 2015 Mar 31 pii gav013 [Epub ahead of print]
Chromosomal meiotic segregation embryonic developmental kinetics
and DNA (hydroxy)methylation analysis consolidate the safety of
human oocyte vitrification De Munck N Petrussa L Verheyen G Staessen C Vandeskelde Y Sterckx J Bocken
G Jacobs K Stoop D De Rycke M Van de Velde H
Hum Reprod 2013 Aug28(8)2101-10 doi 101093humrepdet107 Epub 2013 Apr 16
Lower intracellular concentration of cryoprotectants after
vitrification than after slow freezing despite exposure to higher
concentration of cryoprotectant solutions Vanderzwalmen P1 Connan D Grobet L Wirleitner B Remy B Vanderzwalmen S
Zech N Ectors FJ
Safety of Vitrification
bull Fully open system theoretical risk of contamination is
gt500000 transfers
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Which embryos do you freeze
bull Every lab has their own criteria
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Increase in Freeze All Cycleshellipwhy
bull PGS has something to do with this
bull Research to show that transferring embryos in an non-stimulated
cycle improves outcomes for mother and baby
ndash singleton pregnancies following frozen versus fresh embryo
transfers were significantly less likely to be complicated by
bull perinatal mortality [relative risk (RR) 068 (95 confidence
interval (95CI) 048ndash096)]
bull small for gestational age [RR 045 (95CI 030ndash066)]
bull preterm birth [RR 084 (95CI 078ndash090)]
bull low birthweight [RR 069 (95CI 062ndash076)]
bull antepartum haemorrhage [RR 067(95CI 055ndash081)]
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Oocyte Cryopreservation
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Ovarian Tissue
bull Feasible fertility preservation alternative to oocyte freezing
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
3 different types of genetic testing
bull Aneuploidy screening ndash 24 chromosome screening
ndash SNP array CGH NGS
bull Chromosome rearrangement testing
ndash SNP array CGH NGS or FISH
bull Single gene testing
ndash Single gene disorders eg Cystic Fibrosis SMA Huntingtons ndash Test for genetic mutation and transfer unaffected embryos
ndash Karyomapping PCR or SNP+aneuploidy
PGDPGS
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Embryo Biopsy
Day 3
Blastocyst
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Day 3 Embryo biopsy
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Blastocyst biopsy
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
bull Day 3 biopsy
- Day 3 embryos show high levels of chromosome abnormality
and mosaicism
- Biopsy of one cell may not be representative of the
remainder of the embryo
- Only one cell to analyse
bull Blastocyst biopsy has been associated with
- Higher implantation rate than Day 3
- Lower aneuploidy rate than Day 3
- High implantation rates regardless of maternal age
- Reduced mosaicism compared with Day 3
- More cells available for analysis
Day 3 or Blastocyst Biopsy
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Aneuploidy difference according to stage
bull Blastocysts display significantly lower aneuploidy rates
bull Aneuploidy rate continues to decline through pregnancy
Day 3 ~20-25
euploid
Day 5 ~50 euploid
Spontaneous abortion
~50 aneuploid Stillbirth
~4 aneuploid
Livebirth
~03 aneuploid
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Rsup2 = 09724
0
10
20
30
40
50
60
70
80
90
100
lt34 34-35 36-37 38-39 40-41 42-45
Blastocyst Euploidy Rate by Age
Euploidy rate () Linear (Euploidy rate ())
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
Day 3 versus Day 5 biopsy
24 chromosome screening data
Age Day 3 Day 5
Implantation
()
Aneuploidy
()
Implantation
()
Aneuploidy
()
30-34 517 485 632 342
35-39 401 645 607 450
40-42 324 790 615 650
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
24 chromosome screening
Why do it
Which embryo should be
transferred
How many transfers would
be needed to select the right
one
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you
With the knowledge we have today we do all we can
do to assure the health of our patients the babies
resulting from our programs and the adults they
will become
Thank you