Prediction of gamete/embryo viability by birefringence imaging

Markus Montag, Ph.D., Prof.University of Heidelberg, markus.montag@med.uni-heidelberg.de

The principle of polarization microscopy

• Visualizesstructureswithbirefringentproperties

Polarizer

Analyzer

History of polarization microscopy

• 1808: Malus discovers „Polarization“• 1834: 1st 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: 1st commercial polarization microscope system

that can be used with oocytes

Sperm head birefringence

Sperm head birefringence

Looking at oocytes

When is the spindle visible

pH of the mediumTemperature• Critical temperature: 33oC

• 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. 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

Publications on the prognostic value of spindle imaging

Spindle visible Spindle not visible

Fertilization 4 x ↑ 2 x Ø

Embryo dev. day 3 5 x ↑ 2 x Ø

Blastocyste rate 3 x ↑

Preg.-/Impl.-rate 2 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

75-90 minMII

40-60 min

Transition from telophase to metaphase-II

? Is this relevant ?

• ICSI patient, 7 oocytes after puncture after denudation 6 h later

3 0

2 0

2 0

0 7• 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 nm 1.8 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 inmetaphase-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 pellucidaBirefringence of the zona pellucida

Inner zona layer

Middle and outerZona layer

Zona-imaging – a possible parameterfor cytoplasmic maturity?

Zonaimage – Cytoplasmic MaturityZona = product of the oocyte Optimal maturation = good structured zona

Scoring examples

+high

-low

+/-intermediate

Zona imaging: clinical use

0,0

10,0

20,0

30,0

40,0

50,0

60,0

70,0

HZB/HZB HZB/LZB LZB/LZB Kontrolle

*#

% 20 50 65 68 cycles

LZB/LZB versus HZB/LZB and HZB/HZB* P < 0.005# P < 0.025

Pregnancy rate

Implantation rate

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

87,5

63,3

0

10

20

30

40

50

60

70

80

90

%

High Low

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., 2007/8 Prospective Impl./Preg.-rate

Embryo development• Ebner et al., 2009 Prospective Blastocyste formation• Madaschi et al., 2009 Prospective Impl./Preg.-rate

Abortion rate• Cheng et al., 2010 Retrospective No clear effect in IVF• Gu et al., 2010 Retrospective 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: 7.6Mean 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)

-20

0

20

40

Arb

itra

ry Z

on

a s

co

re

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 Zona score and cumulus gene expressionexpression

mRNA expression in corona-radiata-cells

0

2

4

6

8

10

12

BMP-15 CX-43 STAR

Low Score

High Score

P=0.031

N.S.

N.S.

Zona imaging:the cumulus background

Pregnancy No 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 3 Day 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!