Time lapse observations of pre-implantation embryos

Giles Palmer, Mitera ACU, Athensgpalmer@mitera.gr

There are no commercial relationships or other activities that might be perceived as a

potential conflict of interest

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

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

Embryo selection• Invasive techniques

Euploid selection (PGS) not fulfilled expectationsCGH replacing FISHRCT underway

• Non-invasive techniques-”omics” search for biomarkers: metabolomics-not implemented at present. Promising developments but time consuming/ technically challenging

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

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

Static observations are misleading!

Fragmentation:•Pribenszky (2010)12% embryos fragmenting89% reabsorbedAverage time for fragments to appear/disappear 9.1 h ( +/- 442) Chance for NOT noticing fragments at bi-daily monitoring: 72% !!!

Blastocyst contractions:

videos

Which embryo would you transfer?

Static observations are misleading!

Ww

26:32 29:52 30:02

35:12 42:02 42:32

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

5-8cc30-50 min

9-16cc40-70

min

3-4cc10-20min

8-9cc22-24

hrs

4-5cc14-16

hrs

2-3cc10-12

hrs

Interphase 1-2cc

20-26 hrs

Exact timing of interphases

1st cycle 2nd cycle 3rd cycle 4th cycle

Hlinka 2010

Cell CycleCellular organization Nuclear organization and MitosisCell divisionCell synchronicity

Karyokinetics and cytokinetics

Karyokinetics and cytokinetics

Karyokinetics and cytokinetics

Second cell cycle

Third cell cycle

3-4 cell

5-8 cell

t2 t3 t4 t5

cc2 cc3s2

t: exact timet2: time to 2-cellt3: time to 4-cellt4: time to 4-cellt5: time to 5-cell

cc: cell cyclecc2 = t3-t2cc3 = t5-t4

S:synchronys2 = 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

t5

Hierarchical classificationDiscard?

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

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

HDHDGHGDGHHHHHHHHHHHHHHHJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

Abnormal cell division & aneuploidy

Time line profile

e

Time line profile

e

eCGH profile- molecular karyotype

3-4 CellsAbnormal cell division & aneuploidy

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 CGHThe timing of first and second divisions were delayed in aneuploidy and more marked in those with multiple aneuploidy2-4 cell transition: euploidy<single aneuploidy<multiple aneuploidy

Basile (2013) Increasing the probability of selecting chromosomally normal embryos by studying their kineticsClassification 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 PGSAneuploidy risk model

Abnormal cell division linked to aneuploidy

The era of morphokinetics

Chen 2013

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

Cell CycleNuclear organization and MitosisCellular organization Cell divisionCell synchronicity

Zygotic ClockMaternal to zygotic

Fertilization

Maternal control Zygotic control

Karyokinetics and cytokinetics

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%)