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 • Blas tocyst bi opsy • 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 • Reduced uterine contractabilit y 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 100 Maternal mRNA 80 Embryonic mRNA 60 40 mRNA (ng/embryo) mRNA 20 25 4050 60 70 90 110 140 Hrs Fertilization 24 816 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 ICM development • Ménézo 1992 A: tightly packed, many • Gardner 1999 cells B: loosely grouped, several Scoring system cells C: Very few cells Degree of expansion and hatching status Trophectoderm 1.-early blastocyst development 2.- young blastocyst A: many cells forming a 3.-full blastocyst cohesive epithelium 4.-expanded blastocyst B: few cells forming a loose 5.-hatching blastocyst epithelium 6.-hatched blastocyst 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. To help protect your privacy, PowerPoint prevented this external picture from being automatically downloaded. To download and display this picture, click Options in the Message Bar, and then click Enable external content. 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 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 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 • X aggye 38.5 years • 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, implantation failures. Age? Failure to reach embryo transfer (minimal ovarian response?) • Cumulative pregnancy rate (fewer embryos cryopreserved) • Monozygotic twinning THANK YOU FOR YOUR ATTENTION! [email protected] [email protected].