Time-lapse

- what have we learned

Markus Montag

Disclosure

This speaker has received during the last five years honorary / sponsoring from the following companies in alphabetical order:

- Cook, Ferring Arzneimittel GmbH, Gynemed, - Medical Technology GmbH, Merck Serono, - Merck Sharp & Dome, OvaScience, FertiliTech

- This speaker is CEO of ilabcomm GmbH, a consultancy company,

- A major contractor of ilabcomm GmbH is FertiliTech

Traditional embryo evaluation

68 hrs 44 hrs 25-27 hrs 17-19 hrs

>1500 images over 3 days per embryo

Time-lapse monitoring

68 hrs 44 hrs 25-27 hrs 17-19 hrs

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We move from 1 observation / day

to continuos observation

Time-lapse has entered the clinical

routine only in recent years

J Cohen ALPHA Keynote lecture 2012

Time-lapse in the scientific community-

Increasing number of published data*

*Abstracts / publications based on the EmbryoScope® time-lapse system

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Peerreviewedar cles

ASRMAbstracts

ESHREAbstracts

What have we learned

Explaining controversial issues

Showing variation in PN scoring

17.3h 22.7h 13.4h 17.7h

17.3h 19.0h

Montag et al., 2011

Early cleavage:

with time-lapse we move from

• Yes or no –”do we have early cleavage or not at 26 h PI”

to

• Know the exact time of when the embryo cleave

Clinical impact of time-lapse on

early cleavage: explaining controversy

IVF n = 2091 embryos ICSI n = 5838 embryos

t2

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Multicenter database evaluation; Montag, Petersen et al.

New insight on fragmentation:

fragments can be re-absorbed

Hardarson T el al 2002, RBMOnline and Lemmen et al. 2008, RBMonline

New selection criteria

Early timings

Pronuclear breakdown and live birth

• Azzarello et al. studied prospectively the time of PN breakdown and the PN score in 159 embryos which were all later transferred.

• The PN morphology in the 46 which resulted in live birth was compared with the 113 embryos which resulted in no live birth

PN breakdown and live birth

• PN breakdown occurred never earlier than 20 h and 45 min in embryo resulting in live birth significantly later (significant compared to no live birth).

• Z score had no effect in predicting implantation

Azzarello et al., 2012 Human Reproduction

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Optimal range

Out of range

t3

Working with quartiles

Exact timing of main embryo

development events Establishing optimal ranges

Two cells

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t3

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t5

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Four Cells

t4

In and out of range

• Significantly improved clinical pregnancy rate in a retrospective study by a relative increase of 20% – P value 0.0043

– Odds ratio 1.201 (CL 95% 1.059-1.362)

– Meseguer et al., Fertil Steril 2012

• A prospective randomized trial confirmed the results from the retrospective study – Meseguer et al., submitted

– abstracts presented at ACE2014 / ASPIRE 2014

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EmbryoScope

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The clinical impact of applying a time-

lapse strategy in the clinical routine

*EmbryoScopeTM

But we also learned that timings are

variable and differ between clinics

Evaluation of published data on

timing of cell divisions

Chamanyou S et al. 2013. J Assist Reprod Genet

Timing and implantation for the

duration of the 8-cell stage

Chamanyou S et al. 2013. J Assist Reprod Genet

Knowing the exact timing for a given

clinic is important

t8 - hourly steps

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Distribution of t8 (bars) and implantation rates (curve)

Reduced O2, n=1974; Montag et al., ASPIRE 2014

New de-selection criteria

Cleavage patterns: what we expect

• Normal cleavage pattern

Cleavage patterns: what we can see

• Unusual cleavage patterns

1-3

2-6

2-5 2-(4)-6

2-(4)-5

2-(3)-5

Abnormal reverse cleavage

Cleavage from 1-blastomere to 3-blastomeres in less than 5 hrs

Direct division from 1- to 3-cells

N=109 N=1550

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1, 1% Direct 1->3 division

hCG neg

Biochemical abortion

Clinical abortion

Partial implantation

Known implantation

804, 52%

94, 6%

82, 5%

367, 24%

203, 13%

Not direct 1->3 division

Rubio I et al. 2012Fertility and Sterility / retrospective observational multicenter study

Timings and division charts do

reveal abnormal development

Normal dividing embryo

Embryo proceeding directly from 1 to 3 cells and 3 to 5 cells.

Time-lapse reveals more

multinucleated embryos

26.2 18 65,9 44.6

119.8 105.3

31.9

4-10 cell embryo with multinucleation

Slides courtesy by Dagan Wells / Wells et al., Hum. Reprod. & Fertil. Steril. (2005)

Gene expression, stage & multinucleation

Pre-genome activation 2-cells, 3-cells, early 4-cells

Oocytes

Morulae

Blastocysts

(hatching)

2-10 cell embryos

BRCA1- Roles in repair of

DNA damage (chromosome breakage) and chromosome segregation

The clinical impact: How much do we see with

standard (static) evaluation* versus time-lapse

Direct divison 1- to 3-cells

Multinucleation at the

2-cell stage

MN at the 4-cell stage

Division 2- to 5-cells

Extremely low implantation potential –

missed 63% at 24-26 hrs

40% reduction implantation potential –

missed 21% at 44-45 hrs

Various reports of reduced implantation potential–

missed 23% at 44-45 hrs

Various reports of reduced implantation potential–

missed 38% at 24-26 hrs

* Using the timings recommended in the Alpha / ESHRE consensus

External factors affecting

the embryo timing

Factors affecting morphokinetics…

t2 t3 t4 t5 t8

cc1 cc2 cc3

s2 s3

Age

ICSI Media

pH Oxygen

Aneuploidy Viability?

Etiology

Temperature

Handling

Embryo Development

Stimulation

Maturity

Biopsy

Smoking and embryo quality

Freyour T et al 2012: Oral presentation ESHRE 2012

Fertility and Sterility® Vol. 99, No. 7, June 2013

Results :

- No difference in conventional morphology

- Significant difference in the kinetics of cell division:

- Embryos of smoking women reached the 3-cell and 5-cell stage

slower than the embryos of non-smoking women (p= 0.01)

Oxygen concentration

Objective: “To evaluate, using time-lapse monitoring, the temporal influence of culture in 5% O2 or 20% O2 on human embryonic development”

Retrospective observational cohort study

Group 1 120 embryos

IVF/ICSI 20% O2 (ES)

Group 2 123 embryos

IVF 20% O2 (18h), 5% O2 (ES)

Goup 3 120 embryos

ICSI 5% O2 (ES)

Culture to blastocyst – morphokinetic analysis – no implantation data

Kirkegaard et al. (2013);Fertility&Sterility, 99(3)

Culture Conditions (O2) Kirkegaard et al. (2013);Fertility&Sterility, 99(3)

• Severely delayed timing at late cleavage stages in ambint air • Already an exposure for 18 hours has an effect (Group 2)

t5 - hourly steps

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Comparing t5 timings and implantation rates between reduced O2 (blue bars, solid line) and ambient O2 (hatched bars, dashed line), n=5365

Impact of Oxygen on t5

Applying time-lapse in

quality control

QC: Morphokinetics versus MEA - Toxic Oils – Wolff et al., Hum. Reprod. 2013

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Morphokinetic Model Blastocysts

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Control

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Slide courtesy from Dean Morbeck, Mayo Clinic

The next step forward :

Applying models / algorithm for evaluating

the implantation potential of embryos

in the daily routine clinical work

Potential selection parameters

have been identified

• Correct fertilization*

• Optimal ranges for the timing of cleavages*

• Optimal division characteristics*

• Optimal morphology

– blastomer size variation

– fragmentation etc.

*Scientific evidence available either retrospective observational or prospective

Potential de-selection parameters

have been identified

• Fast division like 1 to 3, 2-5*

• Multinucleation

• Reverse cleavage

• Uneven blastomere size 2-cell / 4-cell*

*Scientific evidence available either retrospective observational or prospective

Models: from basic to tailor-made including clinic characteristics

15.000

Meseguer et al., 2011, Human Reproduction 26:2658-71

0% versus 100% Implantation

Selection marker:

1.Division to 5-cell embryo: 48.8-56.6 h (meanwhile corrected 43-57 h)

2. Time in 3-cell stage: < 0.75 h

3.Division to 2-cell embryo: 24.6-28.2 h

Potent models have been published

- but need adaption to clinics

• Established with ambient air (no low oxygen)

• Numerous cycles with donated oocytes

The current problem with models

- Clinic A vs B / Day 3 vs 5

Time or cell cycle inteval

Clinic B – implantation potential Clinic A –implantation potential

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Optimal range clinic A

Optimal range clinic A applied at clinic B

Conclusion

• Time-lapse has arrived and it will change our way of embryo assessmenct

• Time-lapse has already today a clinical impact

• New selection criteria have been found and introduced:

– PN fading

– Exact time for t2 and t8 is important

– Cell cycle lenght have an impact

– Fast division cycles

– Time to reach the blastocyst

Thank you for your attention ?