blastocyst transfer - eshre
TRANSCRIPT
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
stageBl t t bi• Blastocyst biopsy
• 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 embryo-endometrial synchronicity
Red ced terine contractabilit• Reduced uterine contractability on day 5-7
• In vitro embryo selection. Highest implantation potential?
• PGD programmes
• Reduction of multiple pregnancies by single blastocyst transfer
• Diagnostic tool
BLASTOCYST CULTUREBLASTOCYST MORPHOLOGY AND QUALITY ASSESSMENT
QE
Maternal mRNA
Embryonic mRNA
mR
NA
(n
g/em
bry
o)
20
40
60
80
100
25
8 162 4Fertilization
40 60 70
Stage
50 90 110 140 Hrs
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 improvedthe blastulation rate and increasedthe % of embryos reaching the stageof expanded blastocysts with normal ICM on day 5
• The ratio for successful developmentto optimal blastocyst stage is 2.1 forIVF and 1.7 for ICSI in favour of lowerO2 tension
• The overall increase in livebirths indicates thatthe effort and expense toculture embryos in low O2 environment is justified.
• Ménézo 1992• Gardner 1999
Scoring system
Blastocyst scoring
ICM developmentA: tightly packed, many cellsB: loosely grouped, several cellsC: Very few cells
Degree of expansion and hatching status1.-early blastocyst2.- young blastocyst3.-full blastocyst4.-expanded blastocyst5.-hatching blastocyst6.-hatched blastocyst
y
Trophectoderm developmentA: many cells forming a cohesive epitheliumB: few cells forming a loose epitheliumC: 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 blastocystselection
• TE important for successfullhatching and implantation
• NIR spectroscopy does not improve the chance of a viable pregnancy whenchance of a viable pregnancy when performing SET
• Further developement of the technology is needed.
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• Success in progression to the blastocyst stage can be predicted with >93%
NATURE BIOTECHNOLOGY 2010
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 thatanalysis reveals that blastomeres develop cell autonomously, with some cells advancing to EGA and others arresting.
Day 3 vs D 5 transferWhen to go for blastocyst transfer?When to go for blastocyst transfer?
When to go for blastocyst transfer?
P<0.006
Racowsky et al., 2000
n.s.
P 0.006
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
• 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
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.
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 transferStern et al, Fertil Steril 2008
BLASTOCYST AND ESET
The findings support the transfer of single
2006
gblastocyst-stage (day 5) embryo in women under 36 years of age
Fertil Steril 2007
Decreased twin rates with a mandatory single blastocyst transfer policy
• 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 2008
eSET at the blastocyst stage in good prognosis patients
Fertil Steril, 2009
prognosis patients reduces twin pregnancies wthout compromising pregnancy rates.
A novel single-blastocystalgorithm reduced multiplegestation rates and improved
Fertil Steril 2011
gestation rates and improvedcryopreservation rates withoutcompromising clinical pregnancyrates in good-prognosis patients
CHROMOSOMAL ABNORMALITIES AT THE
BLASTOCYST STAGEBLASTOCYST STAGE
Aneuploidy selection: D3 vs Blastocyst
Staessen et al., 2004
• X age 38.5 yearsg y
• 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?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
• Provides more cells to analyse
• Interesting in monogenic diseases (more DNA
available)
• Lower degree of mosaicism
Blastocyst biopsy
• 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 toidentify 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
• Male embryos do not
Fertil Steril 2009
grow faster thanfemale embryos in vitro
• No sex ratio imbalance is observedin 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 with low quality scores are able to give rise to hESC lines even the efficiency is low
embryos)
•Chen et al, Hum Reprod 2005
•Kim et al, Stem Cells 2005 (derivation method depending on blastocyst quality)
In Vitro Cell.Dev.Biol., 2010
BLASTOCYST FREEZING
• Cryopreserved day 5 blastocysts have higher implantation rates than day 6 blastocysts
• Acceptable outcomes with day 6 blastocysts
Fert Steril 2008
The feasible strategy in good responder patients is the cryopreservatiopn of blastocysts
Fert Steril,2011
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 implantation failures• Indications: All patients, implantation failures. Age? Failure to reach embryo transfer (minimal ovarian response?)
• Cumulative pregnancy rate (fewer embryos cryopreserved)
• Monozygotic twinning
