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Oocyte and Embryo Selection using Sequential Embryo Selection ( SES )

Oocyte and Embryo Selection using Sequential Embryo Selection ( SES ). Lynette Scott Fertility Centers of New England Reading, MA, USA. Gametes. Abnormal gametes generally do not produce normal embryos. Early embryo parameters may be a window back to the gametes. Day 1 and Day 2. Day 3.

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Oocyte and Embryo Selection using Sequential Embryo Selection ( SES )

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  1. Oocyte and Embryo SelectionusingSequential Embryo Selection (SES) Lynette Scott Fertility Centers of New England Reading, MA, USA

  2. Gametes Abnormal gametes generally do not produce normal embryos Early embryo parameters may be a window back to the gametes Day 1 and Day 2 Day 3 Day 5 Later development reflects gene expression, differentiation, developmental controls Differentiation

  3. Human Oocyte Cumulus Cells -NO -gene up/down regulation - Oocyte -Mt load -cAMP -nuclear/ Cytopasmic maturation

  4. Zona Pellucida • Laid down by the oocyte so could reflect oocyte quality • Can be visualized and measured using polarized light microscopy • 2 systems: A- measures zona density B- measures differential zona layers and thickness

  5. Porous surface of the zona pellucida

  6. Zona Density, Alignment, Abnormalities Courtesy of Marcus Montag Layers Alignment

  7. Spindle • Visualized using polarized light microscopy • Position- should be in the hemisphere containing the polar body • The shape of the spindle is more important • Should be bi-polar and ordered • Correlations with oocyte competence • Draw backs: • 98% of spindles are in the correct position • The spindle is very dynamic and temperature sensitive, forming and dismantling in cycles and with temperature drops. The oocyte must also be very carefully positioned for accurate visualization. • Only true irregularities = abnormal

  8. Metaphase II Spindle Note Spindle Alignment And Zona Alignment Courtesy of Laura Rienzi

  9. Abnormal Spindle Shapes Courtesy of David Keefe

  10. Fertilized Oocyte ScoringLooks at a part of nucleoli in the early embryo Nucleoli • Found in all actively dividing cells • Sites of rRNA synthesis • Develop on the DNA where the genes for ribosomes are located, rDNA • These points on the DNA = NORs • Nucleolar Organizing Regions

  11. Human NORs • 5 pairs of NOR-bearing chromosomes • 13, 14, 15, 21, 22 (acrocentric) • Generally 5-7 NORs in human cells • NOR’s are clustered and this is dependent on heterochromatin adjacent to rDNA genes • Heterochromatin is not inactive and may be involved in developmental control (Dimitri, 2004) • Transcription of rDNA results in 3 functional parts:

  12. Nucleolar Precursor Body (NPB) Pattern Normal = equality between nuclei Abnormal = any inequality

  13. NPB RATIO L = 4 R = 4 L = 5 R = 6

  14. P<0.05 NPB Ratio vs. Outcome

  15. Condensed chromatin 13, 14, 15, 21, 22 NPB RNA Polymerase Fibrillar component and center Pre-RNA Dense fibrillar component Granular component

  16. NPB IN FERTILIZED OOCYTES Looking at chromatin 13, 14, 15, 21, 22 NPB L = 5 R = 6

  17. Evidence for epigenetic/ imprinting errors in these oocytes Sperm or oocyte? NPBs associated with imprinting errors, heterochromatin

  18. 34 yr old, GO, PO Sever male factor, Sperm problem not an egg one 6 IVF attempts, no pregnancy, 2 PGD, no normal’s 1 DI attempt, Term delivery, 3628 gr male

  19. Day 2 Scoring • Cell number • Blastomere relative size • Status of nucleation • Fragmentation • Timing is important

  20. Day 2 Cell SizeWhy is it important? • Polarity • Distribution of cell components • Embryo axes • The meiotic and mitotic spindle

  21. 30% MULTI-NUCLEATION and CELL SIZE

  22. 30% Small Rosette 4-cell Blastomer Morphology

  23. Day 2 Polyploid Complex Abnormal Day 3 Day 1

  24. Day 1 and Day 2 Correlations

  25. Deliveries according to early morphometrics Scott et al., 2007

  26. Day 3 Cell Number P<0.05

  27. Early Scoring Parameters Significant for Delivery Scott et al., 2007

  28. Early Scores and Delivery Scott et al. 2007

  29. Complex Abnormal XXXY 1x 16 1x 21 3x 22 0x 15 MN MN Good Grade 8-cell Good Blastocyst (D6)

  30. SEQUENTIAL SELECTION • Accept that “pretty” good • Use biologic criteria and not only morphology • In gated scoring, an embryo that does not pass through the first gate must not be selected at the second level, regardless of morphology • If it does not pass the 2nd gate it should be discarded, as this is the most NB criteria

  31. Introduction and use of SESImpact on Delivery Rates 1.9* 2.1 2.4 2.1 2.2 2.5 % Delivery/>20 weeks 5% 2% 1% * Mean # ET 2.1 Twin Rate 30-40%

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