1. Prediction of gamete/embryo viability by birefringence imaging Markus Montag, Ph.D., Prof. University of Heidelberg, markus.montag@med.uni-heidelberg.de

2. The principle of polarization microscopy Visualizes structures with birefringent properties Polarizer Analyzer

3. History of polarization microscopy 1808: Malus discovers „Polarization“ 1834: 1 st commercial polarization microscope 1875: Engelmann reports on the birefringent properties of frog sperm 1924: Schmidt describes cytoskeletal elements based on polarization microscopy 1953-1981: Inoué / Allen publish numerous ground breaking articles on microtubules and the spindle 1998: 1 st commercial polarization microscope system that can be used with oocytes

4. Sperm head birefringence

6. Looking at oocytes

7. When is the spindle visible pH of the medium Temperature Critical temperature: 33 o C Culture medium Heated surfaces: Stereo microscope Injection microscope Incubator

8. Where is the spindle Spindle at ICSI – First report that spindle is not always where it should be: Silva et al., 1999 – Report that visualization of the spindle position has no benefit at all: Woodward et al., 2008

9. Where is the spindle Spindle- / polar body-relation Rienzi et al. 2003 – Negative effect of fertilization if PB and spindle are dislocated, but no effect on embryo development Cooke et al., 2003 – Positive effect if PB and spindle are not dislocated on embryo development Taylor et al., 2008 – Dislocated PB are the result of a too harsh pipetting mode during hyaluronidase preparation Polar bodies do move: Scott, 2008

10. Publications on the prognostic value of spindle imaging Spindle visibleSpindle not visible Fertilization4 x ↑2 x Ø Embryo dev. day 35 x ↑2 x Ø Blastocyste rate3 x ↑ Preg.-/Impl.-rate2 x ↑1 x Ø More information needed: Petersen et al., 2009 RBMOnline

11. Course of the first meiotic-cycle

12. Course of the meiotic cycle cont. Montag, Schimming, van der Ven; RBM Online 2006

13. Spindle dynamics: a physiological process Spindle bridge MI -> MII 75-90 min MII 40-60 min Transition from telophase to metaphase-II

14. ? Is this relevant ? ICSI patient, 7 oocytes after puncture after denudation 6 h later 30 2 0 07 ICSI: all 7 oocytes fertilized with 2 PN MI MII Spindle bridge MI -> MII

15. Oocyte maturity: how to define Metaphase-II-oocyte (M-II)

16. Relevance for ICSI

17. Spindle presence in relation to [t] hCG Kilani et al., Zygote 2010

18. Spindle shape and success rates Spindle shape may have an impact on success rates BUT Spindle shape is dependent on: – Timing of spindle imaging – Orientation of the spindle Kilani et al., F&S 2011

19. Spindle-retardance measurement in oocytes

20. 1.7 nm1.8 nm 1.4 nm 1.7 nm 1.3 nm At different orientations retardance of a metaphase-II-spindle shows variations ranging from +6% to -20% in relation to a reference value of 1.7nm Spindle retardance measurement in metaphase-II-oocytes: position effect

21. Summarizing spindle imaging Spindle-Imaging – Localisation during ICSI (Silva et al. 1999) – Enucleation (Liu et al. 2000) – Spindle-/polar body relation (Rienzi et al. 2003) – Chromatin aberration (Wang et al. 2003) Spindle dynamics – Temperature sensitivity (Wang et al. 2001) – Meiotic cell cycle (Montag et al. 2006)

22. Birefringence of the zona pellucida Inner zona layer Middle and outer Zona layer

23. Zona-imaging – a possible parameter for cytoplasmic maturity? Zonaimage – Cytoplasmic Maturity Zona = product of the oocyte Optimal maturation = good structured zona

24. Scoring examples + high - low +/- intermediate

25. Zona imaging: clinical use * # % 20 506568cycles LZB/LZB versus HZB/LZB and HZB/HZB * P < 0.005 # P < 0.025 Pregnancy rate Implantation rate

26. Oocyte zona score and embryo - development: 4 cell grade A (d2) to 8 cell grade A (d3) P < 0.05

27. Correlation Zona-Image and ART Shen et al., 2005 Retrospective Preg.-rate Raju et al., 2007 Retrospective Embyo development Blastocyste formation Montag et al., 2007/8Prospective Impl./Preg.-rate Embryo development Ebner et al., 2009 Prospective Blastocyste formation Madaschi et al., 2009Prospective Impl./Preg.-rate Abortion rate Cheng et al., 2010Retrospective No clear effect in IVF Gu et al., 2010Retrospective IVF+ICSI, fresh + frozen- thawed embryos on day 3 No correlation of zona birefringence with embryo development/clinical results

28. What we should keep in mind Mean no. of MII-oocytes retrieved:7.6 Mean no. of HZB oocytes: 1.5

29. Zona score of oocytes from small follicles A high zona score is beneficial BUT The origin of the oocyte must be taken into account M-II oocytes from small follicles show a higher zona score compared to oocytes from medium/large follicles (P < 0.05) Small Med./Large P < 0.05

30. Zona imaging and the source of the oocytes - GV and MI oocytes give generally higher zona scores compared to MII oocytes - MII oocytes from small follicles give higher zona scores compared to MII oocytes from medium/large follicles - If one does not know this background zona imaging will preferably select MII oocytes which are originating from smaller follicles

31. Zona score and cumulus gene expression P=0.031 N.S.

32. Zona imaging: the cumulus background PregnancyNo pregnancy Seven significantly differential expressed genes in cumulus cells Assidi, Montag, van der Ven, Sirard; JARG, 2011

33. Zona imaging during embryo development The zona score rises from day 3 to day 5 – Linked to expansion of the blastocysts and the related changes in the order of the zona Day 0 Day 3Day 5

34. Zona imaging during embryo development Implanted Non-impl. Score in expanded blastocysts, top grade on day 5

35. Summary Spindle imaging is useful – To assess/assure lab quality parameters – In selected cycles with a high rate of immature oocytes to determine the proper time of ICSI Zona imaging is useful – To assist in the identification of oocytes with a high implantation potential – To identify embryos with a better implantation potential Birefringence imaging is an add-on to other prognostic technologies

36. Thank you for your attention!