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

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

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 Courtesy of Laura Rienzi Note Spindle Alignment And Zona Alignment

9. Courtesy of David Keefe Abnormal Spindle Shapes

10. Fertilized Oocyte Scoring Looks 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 Abnormal = any inequality Normal = equality between nuclei

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

14. NPB Ratio vs. Outcome ALLETNot Preg PregFHB100% Implant >12 weeks L/R4/95/84/95/76/7 Range 1-152-10 4-95-95-76-7 P<0.05

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

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

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 Size Why is it important? Polarity Distribution of cell components Embryo axes The meiotic and mitotic spindle

21. MULTI-NUCLEATION and CELL SIZE 30%

22. 4-cell Blastomer Morphology Small 30% Rosette

23. Day 1 Day 3Day 2 Polyploid Complex Abnormal

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

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 SES Impact on Delivery Rates 5% 2% 1% 2.4 1.9 * 2.1 2.2 2.5 2.1 * Mean # ET 2.1 Twin Rate 30-40% % Delivery/>20 weeks