Prediction of gamete/embryo viability by birefringence imaging Markus Montag, Ph.D., Prof. University of Heidelberg,

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

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 : 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

Sperm head birefringence

Looking at oocytes

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

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

Where is the spindle Spindle- / polar body-relation Rienzi et al – 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

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

Course of the first meiotic-cycle

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

Spindle dynamics: a physiological process Spindle bridge MI -> MII min MII min Transition from telophase to metaphase-II

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

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

Relevance for ICSI

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

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

Spindle-retardance measurement in oocytes

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

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)

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

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

Scoring examples + high - low +/- intermediate

Zona imaging: clinical use * # % cycles LZB/LZB versus HZB/LZB and HZB/HZB * P < # P < Pregnancy rate Implantation rate

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

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

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

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

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

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

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

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

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

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

Thank you for your attention!