Timelapseuk

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Morphokinetic studies on in vitro embryo developoment-choosing the best embryo

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  • Embryo graded 1-5 PN on day1 Cleavage patterns Rate of first cleavage Blastocyst development, cell number, rate, inner cell mass aneuploidy
  • Embryo graded 1-5 PN on day1 Cleavage patterns Rate of first cleavage Blastocyst development, cell number, rate, inner cell mass aneuploidy
  • Timelapseuk

    1. 1. Time lapse observations in pre-implantation embryos and factors effecting implantation potential Giles Palmer
    2. 2. ART today <ul><li>1-4% children born European countries </li></ul><ul><li>4000 cycles per year </li></ul><ul><li>22% multiple births </li></ul><ul><li>Success rate rel. low </li></ul><ul><li>Tendency in N. European countries to reduce multiple pregnancies/ SET </li></ul><ul><li>Public health concern </li></ul>Andersen (ESHRE) 2009, Seli 2011
    3. 3. At present <ul><li>Inability to embryo best potential </li></ul><ul><li>Non-invasive-”omics”-promising developments but time consuming/ technically challenging </li></ul><ul><li>Invasive-embryo biopsy-FISH replaced by CGH </li></ul><ul><li>Still relies on Observation </li></ul><ul><li>1-4% children born European countries </li></ul><ul><li>4000 cycles per year </li></ul><ul><li>22% multiple births </li></ul><ul><li>Success rate rel. low </li></ul><ul><li>Tendency in N. European countries to reduce multiple pregnancies/ SET </li></ul><ul><li>Public health concern </li></ul>Andersen (ESHRE) 2009, Seli 2011
    4. 4. Choosing the best embryo!
    5. 5. Elder, 2007
    6. 6. Pronuclear Morphology *
    7. 7. Pronuclear Morphology *
    8. 8. Pronuclei Orientation Balaban. Hum. Reprod. 16.11. (2001)
    9. 9. Pronuclei Orientation Current hypothesis: pronuclei to be Parallel to the polar axis
    10. 10. Different strategies of day of embryo transfer Day 5 Only at blastocyst stage (Higher implantation) Flexible Day 2-3 or 5 What ever the approach- it relies on morphology- difficult to predict 51% Day 5 embryos preselected on day 3 Rijenders. Hum. Reprod. 13.8. (1998 ) Early cleavage
    11. 11. 2010 Scoring System <ul><li>Pronuclear morphology </li></ul><ul><li>Early 1 st division </li></ul><ul><li>Presence of vacuoles, multi-nuclei </li></ul><ul><li>Number of cells/stage </li></ul>Franscovits, 2006
    12. 12. 2010 Scoring System <ul><li>Pronuclear morphology </li></ul><ul><li>Early 1 st division </li></ul><ul><li>Presence of vacuoles, multi-nuclei </li></ul><ul><li>Number of cells/stage </li></ul>Franscovits, 2006
    13. 14. Implantation linked to exact timing events Herrero, 2010 Event PN appear PN fading 1 st division 2 nd division 3 rd division Range (h) 7.8-11.1 Out of range 22.3-25.8 Out of range 24.4-28.2 Out of range 35.3-40.6 Out of range 36.0-41.6 Out of range 100% Implanted N (%) 15 (54%) 13 (46%) 23 (66%) 12 (34%) 23 (66%) 12 (34%) 13 (72%) 5 (28%) 19 (73%) 7 (27%) 0% Implanted N (%) 60 (49%) 62% (51%) 55 (45%) 67 (55%) 57 (46%) 67 (54%) 43 (45%) 52 (55%) 45 (45%) 56 (55%)
    14. 15. Implantation linked to exact timing events Herrero, 2010 Event PN appear PN fading * 1 st division * 2 nd division * 3 rd division * Range (h) 7.8-11.1 Out of range 22.3-25.8 Out of range 24.4-28.2 Out of range 35.3-40.6 Out of range 36.0-41.6 Out of range 100% Implanted N (%) 15 (54%) 13 (46%) 23 (66%) 12 (34%) 23 (66%) 12 (34%) 13 (72%) 5 (28%) 19 (73%) 7 (27%) 0% Implanted N (%) 60 (49%) 62% (51%) 55 (45%) 67 (55%) 57 (46%) 67 (54%) 43 (45%) 52 (55%) 45 (45%) 56 (55%)
    15. 16. Exact timing of interphases ID3 5-8cc 30-50 min ID4 9-16cc 40-70 min ID2 3-4cc 10-20 min IP4 8-9cc 22-24 hrs IP3 4-5cc 14-16 hrs IP2 2-3cc 10-12 hrs IP1 1-2cc 20-26 hrs 1 st cycle 2 nd cycle 3 rd cycle 4 th cycle Hlinka 2010
    16. 17. Cell Cycle Nuclear organization and Mitosis Cellular organization Cell division Cell synchronicity
    17. 18. Cell Cycle Nuclear organization and Mitosis Cellular organization Cell division Cell synchronicity Zygotic Clock Maternal to zygotic Γονιμοποίηση Μητρικός έλεγχος Ζυγωτικός έλεγχος
    18. 19. t2 t3 t4 t5 cc2 cc3 s2 Cc: cell cycle cc2 = t3-t2 cc3 = t5-t4 s2 = t4-t3 cc2=11,8h s2 = <0.76h t2=25,6h (24,3-25,8h) t3=37,4h (35,4-37,8h) t4=38h (36,4-38,9h) t5=52,3h (48,8-56,6,h)
    19. 20. Time lapse <ul><li>>4mm field of view </li></ul><ul><li>1 micron/pixel resolution </li></ul><ul><li>Optimized green light </li></ul><ul><li>1/10000 ambient light </li></ul><ul><li>Robust, micro-engine </li></ul><ul><li>Analysis software </li></ul>
    20. 21. Time lapse <ul><li>>4mm field of view </li></ul><ul><li>1 micron/pixel resolution </li></ul><ul><li>Optimized green light </li></ul><ul><li>1/10000 ambient light </li></ul><ul><li>Robust, micro-engine </li></ul><ul><li>Analysis software </li></ul><ul><li>Housed in incubator </li></ul>
    21. 22. Time lapse
    22. 23. Time lapse <ul><li>Im p roved culture system </li></ul><ul><li>Embryos are cultured in a group ( “ group effect ” ) </li></ul><ul><li>Embryos can develop their own microenvironment </li></ul><ul><li>Embryos ’ cross talk </li></ul>Vajta et al., 2008
    23. 28. Time lapse Parameters
    24. 32. ΜΗΤΕΡΑ _ IVF Results 15 cx, 32-42 yrs., ICSI, Day 2-3 ET Pronuclear appearance/disappearance, cleavage patterns, fragmentation Number Pos B-hCG Total Number of embryo transferred Number of embryos implanted 15 8 (53%) 39 12 (31%)
    25. 33. Fragmentation
    26. 34. In vitro study with mouse e m bryos Prediction of developmental potential, Pribenszky, 2010 Importance of the precise detection of early clea v ages
    27. 35. Reduction of probability of reaching the blastocyst stage if a fragmentation occurs during the embryo development In vitro study with mouse e m bryos Prediction of developmental potential, Pribenszky, 2010
    28. 36. Reduction of probability of reaching the blastocyst stage if a fragmentation occurs during the embryo development In vitro study with mouse e m bryos Prediction of developmental potential, Pribenszky, 2010
    29. 37. Importance of fragmentations Duration of the appearing/ disappearing fragments <ul><li>12% of the embryos were fragmenting </li></ul><ul><li>89% of the fragments have reabsorbed </li></ul><ul><li>The average time for fragments to appear/disappear was 9.1 h ( +/- 442 ) min (dotted line) </li></ul><ul><li>Chance for NOT noticing a fragment at bi-daily monitoring: 72% !!! ( 61% – 94%) </li></ul>
    30. 38. 1-3 cell division Abnormal Cell division
    31. 39. Abnormal cell division, odd numbers of cells linked to aneuploidy
    32. 40. Time line profile e Time line profile e e CGH profile- molecular karyotype 3-4 Cells
    33. 41. What we have learnt? <ul><li>In conclusion </li></ul><ul><li>Insight into developing embryo </li></ul><ul><li>Allowing morphological analysis and temporal analysis </li></ul><ul><li>Kinetic markers for embryo quality a) early cleavage b) 2,3,5 cell </li></ul><ul><li>Exclusion factors: 1-3 cell division, abnormal timing, fragmentation, multinucleiation </li></ul><ul><li>Future </li></ul><ul><li>Adaption-membrane analysis- predict 90% embryo to blastocyst (Wong, 2010) </li></ul><ul><li>Oxygen consumption-increased consumption at fertilization & first division relates to competence </li></ul><ul><li>Learning tool </li></ul>
    34. 42. What we have learnt? <ul><li>Future </li></ul><ul><li>Adaption-membrane analysis- predict 90% embryo to blastocyst (Wong, 2010) </li></ul><ul><li>Oxygen consumption-increased consumption at fertilization & first division relates to competence (Lopes, 2009) </li></ul><ul><li>Learning tool, DVD, report, remote viewing </li></ul>
    35. 43. What we have learnt? <ul><li>Future </li></ul><ul><li>Adaption-membrane analysis- predict 90% embryo to blastocyst (Wong, 2010) </li></ul><ul><li>Oxygen consumption-increased consumption at fertilization & first division relates to competence (Lopes, 2009) </li></ul><ul><li>Learning tool, DVD, report, remote viewing </li></ul>
    36. 44. What we have learnt? <ul><li>Future </li></ul><ul><li>Adaption-membrane analysis- predict 90% embryo to blastocyst (Wong, 2010) </li></ul><ul><li>Oxygen consumption-increased consumption at fertilization & first division relates to competence (Lopes, 2009) </li></ul><ul><li>Learning tool, DVD, report, remote viewing </li></ul>

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