time lapse and embryo morphokinetics

1. Time lapse observations of
pre-implantation embryos
Giles Palmer, Mitera ACU, Athens
gpalmer@mitera.gr

2. There are no commercial relationships or other
activities that might be perceived as a
potential conflict of interest

3. Time lapse in the IVF Laboratory
• Continuous viewing of embryo development
• Embryo monitored from inside incubator
• Creates archives of embryo development used to select
embryo(s) for embryo transfer

4. Embryo selection
• Embryo selection-
subjective assessment
• Call for consensus of
embryo grading
• Early cleavage, pronuclear
morphology & orientation
limited
• Trend to reduce multiple
births after ART

5. Embryo selection
• Invasive techniques
Euploid selection (PGS) not
fulfilled expectations
CGH replacing FISH
RCT underway
• Non-invasive
techniques-”omics” search for
biomarkers: metabolomics-
not implemented at present.
Promising developments but time
consuming/ technically
challenging

6. The (Short) History of Time lapse
monitoring
• Payne (1997) Preliminary observations on polar body extrusion and
pronuclear formation in human oocytes using time lapse
cinematography
• Pribenszky (2010) Pregnancy achieved by transfer of a single blastocyst
selected by time lapse monitoring
• Wong (2010) Non-invasive imaging of human embryos before
embryonic genome activation predicts development to blastocyst stage
• Meseguer (2011) The use of morphokinetics as a predictor of embryo
implantation

7. Time lapse 2013
• Primo Vision (Bright field)
• Embryoscope (Bright field-self contained incubation)
• Eeva (Dark field, automatic embryo prediction software)
Conventional incubator Time lapse microscope Microwell embryo culture dish with the developing embryos

8. Static observations are misleading!
Fragmentation:
•Pribenszky (2010)
12% embryos fragmenting
89% reabsorbed
Average time for fragments to
appear/disappear 9.1 h ( +/- 442)
Chance for NOT noticing fragments
at bi-daily monitoring: 72% !!!
Blastocyst contractions:

9. videos
Which embryo
would you
transfer?
Static observations are misleading!

10. Ww
26:32 29:52 30:02
35:12 42:02 42:32

15. Implantation is linked to exact
timing events
Event PN appear PN fading* 1st
division* 2nd
division* 3rd
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%)
Herrero, 2010

16. 5-8cc
30-50
min
9-16cc
40-70
min
3-4cc
10-20
min
8-9cc
22-24
hrs
4-5cc
14-16
hrs
2-3cc
10-12
hrs
Interphase
1-2cc
20-26 hrs
Exact timing of interphases
1st
cycle 2nd
cycle 3rd
cycle 4th
cycle
Hlinka 2010

17. Cell Cycle
Cellular organization
Nuclear organization and Mitosis
Cell division
Cell synchronicity
Karyokinetics and cytokinetics

18. Karyokinetics and cytokinetics

19. Karyokinetics and cytokinetics
Second cell cycle
Third cell cycle
3-4 cell
5-8 cell

20. t2 t3 t4 t5
cc2 cc3s2
t: exact time
t2: time to 2-cell
t3: time to 4-cell
t4: time to 4-cell
t5: time to 5-cell
cc: cell cycle
cc2 = t3-t2
cc3 = t5-t4
S:synchrony
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)
Proposed a multivariable model to classify embryos into implantation potential

21. t5
Hierarchical classification
Discard?
Exclude?
s2s2
cc2cc2cc2cc2
A+ A- B+ B- C+ C- D+ D- E F
Accept Discard
ExcludeInclude
Within range Outside range
Outside
range
Outside
range
Within
range
Within
range

22. t5
Hierarchical classification
Discard?
Exclude?
s2s2
cc2cc2cc2cc2
66
%
36
%
29
%
24
%
25
%
10
%
10
%
15
%
8
% F
Accept Discard
ExcludeInclude
Within range Outside range
Outside
range
Outside
range
Within
range
Within
range
Implantation

23. HDHDGHGDGHHHHHHHHHHHHHHH
JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ
JJJJJJJJJJJJJJJJJJ
JJJJJJJJJJJJJJJJ
JJJJJJJJJJJJJJJJJJJJJ
JJJJJJJJJJJJJJJJJJJJJJ
JJJJJJJJJJJJJJJJJJJJJJJJJJ
JJJJJJJJJJJJJJJJJJJJJJJJJJ
JJJJJJJJJJJJJJJJJJJJ

24. Abnormal cell division & aneuploidy

25. Time line profile
e
Time line profile
e
eCGH profile- molecular karyotype
3-4 Cells
Abnormal cell division & aneuploidy

26. Time line profile
e
Time line profile
e
e
3-4 Cells
Davies (ESHRE 2012) Delayed cleavage divisions and prolonged transition between 2-4-cell stages
identified as aneuploid at 8-cell by array CGH
The timing of first and second divisions were delayed in aneuploidy and more marked in those with
multiple aneuploidy
2-4 cell transition: euploidy<single aneuploidy<multiple aneuploidy
Basile (2013) Increasing the probability of selecting chromosomally normal embryos by studying their
kinetics
Classification system based on time parameters relates to selection of euploid embryos
Normal embryos A+: 36,3%, A: 33,9%;B+: 32,0%, B:19,5%;C+:14,3%, C:11,5%;D+: 10,0%, D: 9,0%
Campbell (2013) Retrospective analysis of outcomes after IVF using an aneuploidy risk model derived
from time-lapse imaging without PGS
Aneuploidy risk model
Abnormal cell division linked to aneuploidy

27. The era of morphokinetics
Chen 2013

28. Conclusions
• Improves knowledge of in vitro embryo
development
• Potential to standardize embryo assessment
• Easily incorporated into IVF lab
• Exclude embryos with direct/ abnormal
cleavage, sub-optimum development,
fragmentation, multinucleation
• Improved embryo selection may increase
pregnancy rates

29. Cell Cycle
Nuclear organization and Mitosis
Cellular organization
Cell division
Cell synchronicity
Zygotic Clock
Maternal to zygotic
Fertilization
Maternal control Zygotic control
Karyokinetics and cytokinetics

30. Implantation is linked to exact
timing events
Herrero, 2010
Event PN appear PN fading* 1st
division* 2nd
division* 3rd
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%)