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Prediction of gamete/embryo viability by birefringence imaging
Markus Montag, Ph.D., Prof.University of Heidelberg, [email protected]
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!