Non-invasive methods of embryo selection
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1 Non-invasive methods of embryo selection Liow Swee Lian O & G Partners Fertility Centre Gleneagles Hospital SINGAPORE
2 Introduction More physiological laboratory procedures and culture systems have significantly improved implantation rates Transfer of more than a single embryo in an IVF cycle now presents a high probability of a multiple gestation Single embryo transfer (SET) is mandatory in some countries In most countries, not more than 2 embryos are transferred
3 Embryo selection One of the major challenges for embryologists Selection of embryos with the highest potential to implant Current methods of embryo selection are based on detailed morphological parameters at different stages of development
4 Predictive value of oocyte morphology Human oocyte morphological abnormalities Rienzi L et al (2011) Hum Reprod Update 17(1):
5 Predictive value of oocyte morphology Kilani S (2009) RBM Online 18(5):
6 Predictive value of oocyte morphology Markus M (2008) RBM Online 17(4):
7 CONCLUSIONS: No clear tendency in recent publications to a general increase in predictive value of morphological features was found. These contradicting data underline the importance of more intensive and coordinated research to reach a consensus and fully exploit the predictive potential of morphological examination of human oocytes.
8 Morphological embryo grading systems Pronulcear/zygote stage Cleavage stage Blastocyst stage
9 Configurations for Pronuclear Morphology, Nucleolar Morphology and Polar Body Alignment Gianaroli et al (2003) Fertil Steril 80(2):
10 Predictive value of pronulcear scoring Zygote-score assessment has a limited clinical significance in the choice of the best embryos for transfer Evaluation of embryo quality based on the number of blastomeres, embryo morphological characteristics and grade is more predictive than zygote -score Nicoli et al (2010) Reprod Biol Endocrinol 8:77
11 Embryo grading by analysing blastomere nuclei Moriwaki et al (2004) Hum Reprod 19(1):
12 Conclusion(s): Embryo grading systems are useful in the prediction of embryo implantation. In particular, cell number and embryo grade are more predictive than Z-scores. Therefore, late parameters have a better prognostic value than Z-scores when selecting embryos for transfer. (Fertil Steril 2010;93:658 62
13 Blastocyst grading system Blastocyst development stage Inner cell mass score (quality) Trophectoderm score (quality)
14 Blastocyst development stage Expansion grade Blastocyst development and stage status 1 Blastocoel cavity less than half the volume of the embryo 2 Blastocoel cavity more than half the volume of the embryo 3 Full blastocyst, cavity completely filling the embryo 4 Expanded blastocyst, cavity larger than the embryo, with thinning of the shell 5 Hatching out of the shell 6 Hatched out of the shell
15 Inner cell mass score (quality) ICM grade A B C Inner cell mass quality Many cells, tightly packed Several cells, loosely grouped Very few cells Trophectoderm score TE grade A B C Trophectoderm quality Many cells, forming a cohesive layer Few cells, forming a loose epithelium Very few large cells
16 Blastocyst grading system 4AA Well expanded = 4 Inner cell mass = A Trophectoderm = A 4AB Well expanded = 4 Inner cell mass = A Trophectoderm = B 4BB Well expanded = 4 Inner cell mass = B Trophectoderm = B
17 Embryo Morphokinetics Transfer of early cleavage embryos result in higher implantation and pregnancy rates compared with embryo transfers with delayed division (Van Montfoort et al, 2004) Maybe correlated with higher numbers of cells and better viability compared with latecleaving embryos (Shoukir et al., 1997; Sakkas et al., 1998, 2001;Lundin et al., 2001; Salumets et al., 2001; Fenwick et al., 2002).
18 Embryo morphokinetics Meseguer et al (2011) Hum Reprod 26(10):
19 Meseguer et al (2011) Hum Reprod 26(10):
20 Embryo morphokinetics
21 Implantation failure >70 % of IVF embryos fail to implant (Patrizio & Sakkas, 2009) 1. absence of developmentally competent embryos in an IVF cohort 2. our inability to select competent embryo (s) for transfer
22 Aneuploidy and mosaicism in the human blastocyst Fragouli and Wells (2011) Cytogenet Genome Res
23 The OMICS
24 Proteomics It is proposed that appropriate activation of the embryonic genome, and thus proteome, is a critical determinant of potential developmental competence (Katz-Jaffe and Gardner, 2008)
25 Process flow in SELDI-TOF-MS Surface enhanced laser desorption and time-of-flight mass spectrometry A complex protein mixture is applied to a ProteinChip Nonspecifically bound proteins are washed away Energy-absorbing matrix is applied to all samples, and a laser beam desorbs and ionizes the protein crystallized in the matrix Ions are captured and the mass of each protein is calculated by TOF-MS.
26 Non-invasive assessment of embryos Small molecule proteins in culture medium Protein analysis by SELDI TOF Mass spectrometry viable Non-viable euploid aneuploid
27 Non-invasive assessment of embryos
28 Non-invasive assessment of embryos
29 Non-invasive assessment of embryos
30 Non-invasive assessment of embryos
31 Secretome for embryo viability Biomarkers PAF platelet-activating factor- autocrine factor important for the survival of the embryo during its development (O Neill, 2005) Leptin competent blastocysts secreted higher leptin concentrations into the surrounding medium than arrested embryos (Gonzales et al, 2000) -May have a role in implantation (Cervero et al, 2005) Acrogranin- Promotes blastocyst formation (Diaz-Cueto et al, 2000) Soluble human leukocyte antigen G (shla-g) -May have a role in implantation (Sakkas et al, 2003) - Higher pregnancy rates from shla-g positive embryos (Noci et al, 2005; Sher et al, 2005) Lipocalin -1 -Associated with chromosome aneuploidy (McReynolds et al, 2011) - potential marker for non-invasive aneuploidy screening
32 Embryo protein secretome Protein secretome can be used as a non-invasive screening for aneuploidy in embryos Lipocalin-1 can be a potential biomarker for aneuploidy screening of embryos. Increase in lipocalin-1 in spent culture medium in aneuploid blastocyst McReynolds et al (2011) Fertil Steril 95(8):
33 McReynolds et al (2011) Fertil Steril 95(8):
34 Study of Metabolomics The study of global metabolic profiles in a system (cell, tissue, organism) Systemic study of the unique chemical fingerprints that specific cellular processes leave behind, specifically, the study of their smallmolecule metabolite profiles Systematic analysis of the inventory of the metabolites (small molecule biomarkers) that represent the functional phenotype at the cellular level
35 Metabolome Complete inventory of small molecules, nonproteinaceous compounds Metabolic intermediates, ATP, fatty acids, glucose, cholesterol, hormones, etc. Secondary metabolites
36 Utilization of energy substrates by preimplantation embryos during in vitro development PYRUVATE GLUCOSE MOUSE oocyte MOUSE zygote to 8-cell stage MOUSE 8-cell to blastocyst HUMAN oocyte HUMAN zygote to 8-cell stage HUMAN 8-cell to blastocyst
37 Glucose utilization during different stages of preimplantation embryo in vitro development Martin KL et al (1993) J Reprod Fertil 99:
38 Metabolic profiling in IVF
39 Metabolic profiling in IVF
40 ATP production in in vitro-derived embryos Sturmey and Leese (2003) Reproduction 126:
41 Metabolomics Quiet embryo hypothesis proposed that viable embryos have a quieter metabolism than those which arrest (Leese, 2002) Viable embryo Less molecular/cellular damage Genome Transcriptome Proteome not overly compromised Less O2 and nutrients consumed Less repair Less viable embryo More molecular/cellular damage Genome Transcriptome Proteome compromised More O2 and nutrients consumed Major repair/apoptosis
42 Conclusion Genomics, proteomics and metabolomics have contributed significantly to our understanding of the development of the preimplantation embryos. A tool that is developed from a combination of morphokinetic, proteomic and metabolomic assessments of individual embryos would be valuable in selecting the most viable embryo with highest potential to implant.
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