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BLASTOCYST TRANSFER
Anna Veiga1,2, Gemma Arroyo1
1.-Institut Universitari Dexeus
2.-Centre de Medicine Regenerativa
de Barcelona
• Blastocyst culture, morphology and quality
assessment
• D3 vs D5. When to go for blastocyst transfer?
• Blastocyst and ESET
• Chromosomal abnormalities at the blastocyst
stage
• Blastocyst
Bl t
t biopsy
bi
• Blastocyst transfer and sex ratio
• Blastocyst and hESC derivation
• Blastocyst freezing
• Monozygotic twinning
Blastocyst transfer
• Optimization of culture
conditions: from feeder cells
(Vero system, endometrial cells)
to sequential media to unique
media
• Improvement embryoendometrial synchronicity
• Reduced
Red ced uterine
terine contractability
contractabilit
on day 5-7
• In vitro embryo selection. Highest
implantation potential?
• PGD programmes
• Reduction of multiple
pregnancies by single blastocyst
transfer
• Diagnostic tool
BLASTOCYST CULTURE
BLASTOCYST MORPHOLOGY
AND QUALITY ASSESSMENT
QE
mRNA (ng/embryo)
100
Maternal mRNA
80
Embryonic mRNA
60
40
20
40 50
25
Fertilization
2
60
70
90
8
16
4
110
140 Hrs
Stage
Evolution of maternal and embryonic mRNA in
the Human embryo
• Waves of transcriptional activation start at
2-cell stage human embryos. Also we
identified a hierarchical activation of genes
related with pluripotency.
•We developed HumER, a database of
human preimplantation gene expression.
•
•
Lower O2 concentration improved
the blastulation rate and increased
the % of embryos reaching the stage
of expanded blastocysts with normal
ICM on day 5
The ratio for successful development
to optimal blastocyst stage is 2.1 for
IVF and 1.7 for ICSI in favour of lower
O2 tension
• The
overall increase in
livebirths indicates that
the effort and expense to
culture embryos in low O2
environment is justified.
Blastocyst scoring
•
•
Ménézo 1992
Gardner 1999
Scoring system
Degree of expansion
and hatching status
1.-early blastocyst
2.- young blastocyst
3.-full blastocyst
4.-expanded blastocyst
5.-hatching blastocyst
6.-hatched blastocyst
ICM development
A: tightly packed, many
cells
B: loosely grouped, several
cells
C: Veryy few cells
Trophectoderm
development
A: many cells forming a
cohesive epithelium
B: few cells forming a loose
epithelium
C: very few large cells
Braude
et al,
2006
Future Medicine, Reg. Medicine 2007; 1(6), 739-750
• Blastocyst score
• Predictive strength of TE
grade over ICM for blastocyst
selection
• TE important for successfull
hatching and implantation
• NIR spectroscopy does not improve the
chance of a viable pregnancy when
performing SET
• Further developement of the technology
is needed.
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NATURE BIOTECHNOLOGY 2010
• Success
in progression to
the blastocyst stage can be
predicted with >93%
sensitivity and specificity by
measuring three dynamic,
noninvasive imaging
parameters by day 2 after
fertilization, before
embryonic genome
activation (EGA).
NATURE BIOTECHNOLOGY 2010
•Single-cell gene expression
analysis reveals that
blastomeres develop cell
autonomously, with some cells
advancing to EGA and others
arresting.
Day 3 vs D 5 transfer
When to go for blastocyst transfer?
When to go for blastocyst transfer?
Racowsky et al., 2000
P
0.006
P<0.006
n.s.
Patients with >3 embryos x 8C and transfer at D5 are statistically
younger than D3 patients
When to go for blastocyst transfer?
Racowsky et al., 2000
• The nb of blastocysts, expanded blastocysts and the blastocyst
rate increases with the nb of 8C embryos on day 3
•
•
•
0 e x 8C: pregnancy and IR D3>D5
1-2 e x 8C: no difference D3 vs D5
> 3 e x 8 C: Increased IR on D5
Cochrane Database Syst Rev. 2007 Oct 17;(4):CD002118.
Cleavage stage versus blastocyst stage embryo transfer in
assisted conception.
Blake DA, Farquhar CM, Johnson N, Proctor M.
• This review provides evidence that there is a
significant difference in pregnancy and live
birth rates in favour of blastocyst transfer with
good prognosis patients with high numbers of
eight-cell embryos on Day three being the
most favoured in subgroup for whom there is
no difference in cycle cancellation.
• There is emerging evidence to suggest that in
selected patients, blastocyst culture maybe
applicable for single embryo transfer.
Day 3 vs D5 transfer
Stern et al, Fertil Steril 2008
BLASTOCYST AND ESET
2006
The findings support
g
the transfer of single
blastocyst-stage (day
5) embryo in women
under 36 years of age
Fertil Steril 2007
Decreased twin rates with a
mandatory single blastocyst transfer
policy
Fertil Steril 2008
• Reduction of the twin gestation rate with no
significant compromise of the pregnancy outcome.
• eSBT should be used in young, favourable-prognosis
patients with good quality embryos available
Fertil Steril, 2009
eSET at the blastocyst
stage in good
prognosis patients
reduces twin
pregnancies wthout
compromising
pregnancy rates.
Fertil Steril 2011
A novel single-blastocyst
algorithm reduced multiple
gestation rates and improved
cryopreservation rates without
compromising clinical pregnancy
rates in good-prognosis patients
CHROMOSOMAL
ABNORMALITIES AT THE
BLASTOCYST STAGE
Aneuploidy selection:
D3 vs Blastocyst
Staessen et al., 2004
•
•
•
•
•
g 38.5 yyears
X age
21 % Blastocyst rate
Trisomic embryos reach the blastocyst stage (37%;
p<0.001)
Extensive mosaicism in blastocysts
Monosomies compatible with 3rd trimester development
reach the blastocyst stage (X and 21)
Munne et al 2005
-Mosaicism is the most common abnormality
-Mosaicism correlates with blastocyst quality
-40% of mosacis are abnormal
-Aneuploidy is not related to cleavage dysmorphism
-Trisomies reach the blastocyst stage and beyond
• Early cleavage abnormalities such as mosaicism,
trisomy and polyploidy persist in blastocysts and cannot
be completely screened out by extended culture
eventhough some of them have a detrimental effect in
embryo development
• What is the clinical significance of diploid mosaicism?
• Diploid/tetraploid mosaics may represent a normal
feature in blastocysts.
• It seems that the requirement for embryonic progression
to the blastocyst stage may be a high ratio of normal to
abnormal cells.
BLASTOCYST BIOPSY
Blastocyst biopsy
• Provides more cells to analyse
• Interesting in monogenic diseases (more DNA
available)
• Lower degree of mosaicism
• ICM remains fully intact
• Requires a high blastocyst rate, an optimized
culture system and specific laboratory
expertise
Double selection by genetic diagnosis and culture to blastocyst stage
leads to high pregnancy and implantation rates
Blastocyst biopsy on day 5 and transfer
on day 6
Kokkali et al, 2007
Fertil Steril,2010
•Diagnosis obtained from 93.7% of embryos tested
•Aneuploidy rate: 51.3%
•Ongoing PR per transferred embryo :68.9%
•PR: 82.2%
•IR: 50%
Hum Reprod 2008
The combination of blastocyst biopsy,
microarray gene expression profiling and
DNA fingerprinting is a powerful tool to
identify diagnostic markers of
competence to develop to term.
BLASTOCYST AND SEX RATIO
• More male infants than female infants were born
after blastocyst transfer when transfers were
performed as soon as the blastocyst stage was
reached.
• Faster cleavage rate in male embryos
Fertil Steril 2009
• Male embryos do not
grow faster than
female embryos in vitro
• No sex ratio
imbalance is observed
in the offspring
Fertil Steril, 2009
• Significant sex ratio imbalance after blastocyst
transfer (donor oocytes)
BLASTOCYST AND hESC
DERIVATION
Blastocyst and derivation rate in
relation to embryo origin and quality
•Sjogren et al, RBM online 2004
•Findikli et al, RBM online 2005
•Baharvand et al, Develop. Growth Differ. 2006
Good quality embryos achieve blastocyst stage at a
higher rate and give rise to hESC lines with a higher
eficiency
Blastocyst and derivation rate in
relation to embryo origin and quality
•Sjogren et al, RBM online 2004
•Findikli et al, RBM online 2005
•Mitalipova et al, Stem Cells 2004 (discarded
embryos)
•Chen et al, Hum Reprod 2005
•Kim et al, Stem Cells 2005 (derivation
method depending on blastocyst quality)
Embryos with low quality scores are able to give rise
to hESC lines even the efficiency is low
In Vitro Cell.Dev.Biol., 2010
BLASTOCYST FREEZING
Fert Steril 2008
• Cryopreserved day 5 blastocysts have higher implantation
rates than day 6 blastocysts
• Acceptable outcomes with day 6 blastocysts
Fert Steril,2011
The feasible strategy in good responder patients is the
cryopreservatiopn of blastocysts
MONOZYGOTIC TWINNING
• MZ twinning has been reported in IVF
after AH
• Multicentric study: 199 pregnancies,
10 MZ twinning: 5%
• Increase in MZ twinning after
blastocyst transfer
• Independent predictors of
monochorionic pair: AH,
ICSI and Day 5 transfer
• ICSI and Day 5
synergically increase the
risk of monochorionic
placentation
• Culture conditions: culture media, O2
concentration, time lapse.
• Embryo culture may perturb gene expression.
Epigenetic disturbance?
• Indications: All patients
patients, implantation failures
failures.
Age? Failure to reach embryo transfer (minimal
ovarian response?)
• Cumulative pregnancy rate (fewer embryos
cryopreserved)
• Monozygotic twinning
THANK YOU FOR YOUR ATTENTION!
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