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Time Lapse technology; How effective is it for improving clinical success?

Alberto Tejera PhD Clinical Embriologist

alberto.tejera@ivi.es

4th Biennal Congress of UTD (Society of Assisted Reproduction) Society 25-29 September 2013 Antalya-TURKEY

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“There is a need for more accurate embryo selection in human assisted reproduction…”

Scott et al. 2008

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Why are we interested in this technology? Time-Lapse

Cleavage-stage development is a dynamic process in which embryo morphology may change significantly over a time span

Conventional assessment may not detect subtle differences between individual embryos, such as the time to progress from one cleavage division to the next.

The use of a time-lapse system instrument offers new ways of evaluating embryos with more information to supplement current criteria for embryo selection, and to quantify the exact timing of each cell division.

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EmbryoScopeTM (Unisense FertiliTech, Aarhus, Denmark)

Bi/Tri gas incubator with advanced temperature control. Build-in microscope for acquisition of time-lapse images Image adquisition in multiple focal planes every 15 minutes 6 patients with 12 embryos each = 72 samples

Chamber of incubation

Screen where we can see the development on real time(LIVE) or video

External workstation

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DATA FROM THE EMBRYOSCOPE

Which information is

provided by time lapse for 5 years

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t2 t3 t4 t5

t6 t7 t8

Exact Timing of main embryo development events

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Variable Average

CI 95%

Minimum Maximum Lower

Limit

Upper

Limit

t2 27,50 27,39 27,61 18 71

t3 37,72 37,58 37,86 19 71

t4 40,21 40,06 40,35 21 75

t5 50,54 50,35 50,74 22 80

t6 53,58 53,39 53,76 25 87

t7 56,22 56,01 56,43 27 113

t8 58,59 58,32 58,84 33 112

n = 9530

ICSI ~ 14:00h

D1 17:56h

D2

04:13h

06:43h

17:00h

20:00h

22:43h

D3 01:07h

Exact timing of main embryo development events

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487 couples 4903 oocytes

3630 embryos

885 Day 3

- We select embryos from transfer following morphological criteria

- From transferred embryos we select 467 for detailed analysis:

KID Embryos 100% implantation (n=111). 0% implantation(n=356).

Clinically useful? Kynetics and implantation

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time-lapse (Embryoscope D)

The time for first cleavage was recorded in hours after microinjection and classified in quartiles for all 467 transferred embryos.

Based on single observation !

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>28.2 h

14.8% * (n=17/115)

26.4-28.2 h

29.1% (n=34/118)

24.6-26.4 h

28.8% (n=34/118)

<24.6 h

22% (n=26/117)

p<0.01

time-lapse (Embryoscope D);Implantation

24.6-28.2 h post-ICSI

First division(early cleavage)

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10%

15%

20%

25%

30%

35%

<35 35-38 38-40 >42

% Im

pla

nte

d e

mb

ryo

s

* p<0.05

time-lapse (Embryoscope D); implantation

Timing(3 células)

7%

12%

17%

22%

27%

32%

37%

<36 36-39 39-42 >42

% Im

pla

nted

em

bry

os

Timing(4 células)

32.8 % 30.6%

34.4%

28.1 % 25.8%

8.6% *

20.6% *

11.7% *

* p<0.05

35.2-38.1 h 38.1-40.1 h

32.8 % (n=21/64)

30.6% (n=19/62)

<36.3 h 36.3-39.3 h 39.3-41.8 h

25.8% (n=16/62)

28.1 % (n=18/64)

34.4% (n=21/61)

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Positive

10%

15%

20%

25%

30%

35%

40%

45%

Out of

range

48.8-56.6

Imp

lan

tati

on

P=0,001

3rd division

4 cells

T4

First cleavage

2 cells

t2

2nd division

3 cells

t3

4th division

5 cells

t5

15%

17%

19%

21%

23%

25%

27%

29%

31%

Out of

range

24.6-

28,2

Imp

lan

tati

on

P = 0,043

*

10%

15%

20%

25%

30%

35%

Out of

range

35.2-40.5

Imp

lan

tati

on

P = 0,007

*

*

10%

15%

20%

25%

30%

35%

Out of

range

36,6-41,9

Imp

lan

tati

on

P=0,036

*

time-lapse (Embryoscope D); implantation

Optimal Ranges

From quartiles distribution we defined an optimal range

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Exact timing by time-lapse (Embryoscope D)

Divisions and intervals

• cc1=t2: First cell cycle

• cc2=t3-t2: Second cell cycle, duration of period as 2 cells. •s2=t4-t3: Syncrony in division from 3 to 4 cells

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10%

15%

20%

25%

30%

35%

> 0,75 < 0,75

Imp

lan

tati

on

P = 0,024

*

10%

15%

20%

25%

30%

35%

> 12h < 12hIm

pla

nta

tio

n

P = 0,018

*

CC 2

(t3-t2)

S 2

(t4-t3)

3 cells(t3) 4 cells(t4) 2 cells(t2) 3 cells(t3)

Exact timing by time-lapse (Embryoscope D)

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- Forward step likelihood selection method to determine those most relevant variables related to Implantation; Logistic regression analysis;

·Exact Timing of 5 cell cleavage; OR= 3.311 (CI95% 1.654-6.667)

·Sincrony of second cell cycle; OR= 2.040 (CI95% 1.026-4.067)

48.8-56.6 h

< 0.76 h

t5

S 2

(t4-t3)

Exact timing by time-lapse (Embryoscope D)

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Morphology dynamics exclusion criteria

Direct Division

Blastomere symmetry in first cleavage

Multinucleation

N= 1806 ICSI cycles N= 1646 transferred embryos

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715; 14%

4510; 86%

Incidence rate of direct division

Direct division 1-3cells

No direct division1-3 cells

*

Direct Division

A cleavage from zygote to three blastomeres or a cleavage from zygote to two blastomeres and then to three blastomeres in less than 5 hours.

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2055; 28%

5383; 72%

Uneven

Even

1529; 85%

277; 15% Transferred

Even

Uneven

*P<0.01

Blastomere symmetry in first cleavage

Uneven Even

Imp

lan

tati

on r

ate

11,8%

28,4%

Blastomeres were considered uneven sized if the average diameter of the largest blastomere was more than 25% larger than the average diameter of the small blastomere.

*

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Mn 4 cell stage

*P<0.0001

Multinucleation

*

Embryos were considered MN at the 4 cell stage when more than one distinct nucleus was observed in one (or more) blastomeres.

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Algorithm for embryo selection

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Morphology

included

ok

Grade A Grade B Grade C Grade D Grade E Descarded

excluded

yes no

yes no no yes

A+ A B+ B C+ C D+ D

CC2<12h CC2<12h CC2<12h CC2<12h

yes no yes no yes no yes no

Exclusion

criteria

Direct cleavage

Uneven blastomere

Multinucleation

T5

48,8-56,6h

S2

<0,75h

S2

<0,75h

non viables

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Algorithm; implantation

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P<0.001

Clinical validation

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Blastocyst and ESD Algorithm; blastocyst

40,00%

45,00%

50,00%

55,00%

60,00%

65,00%

70,00%

75,00%

80,00%

A B C D

77,00%

73,00%

65,00%

53,00%

Bla

stoc

isto

(%)

P<0.001

N= 872 % Blastocyst N= 396 Optimal Blastocyst

P<0.001

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*

*

229 477 74 134 14

Morphology dynamics; % blastocyst formation

*

540 12 150

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Kinetics; Exploring novel parameters

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Blastocyst Collapse: Definition

30

40

50

Contracted Not

Contracted

37,6

48,9

Imp

lan

tati

on

Rate

During Blastocyst expansion, embryo is partially or fully collapsed or contracted one or several times prior to transfer.

N= 507 ICSI cycles N= 715 blastocysts transferred

INCIDENCE 19,1% of blastocysts

collapse

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Early compaction

20

30

40

Early Compacted Not Compacted

37,6

26,4

Im

pla

nta

tio

n R

ate

*

N= 3532 couples N= 3782 embryos with KID

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Chromosome content and Time-lapse

5,00%

10,00%

15,00%

20,00%

25,00%

30,00%

35,00%

40,00%

A B C D

35,90%

26,40%

12,10%

9,80%% N

orm

al E

mb

ryos

N= 504 embryos

10%

20%

30%

40%

Out 47.2-58.2

19%

35%

% N

orm

al

Em

bry

os

P=0.003

t5

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- Effect of ESD on clinical pregnancy; OR= 1.201 (CI95%

1.059-1.363) p=0.0043

+ 19 % (IC95% 5.8-37.0%)

9.5%

Is the clinical use of time-lapse system (Embryoscope) improving our clinical results?

+ 19 % (IC95% 5.8-37.0%)

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Hypothesis

More Observations Better Selection

Less Disturbance Better Development

HEPA

particle filter

VOC

carbon filter

UV-light

decontamination Trigas

mixing

>120 L/hr

Air circulation

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Time-Lapse

1. Time-lapse provides markers of embryo viability

2. Clinically available; morphokinetics

3.

Algorithm for embryo selection has been established, but the variables could change, so, each lab should develop its own

algorithm

4. Kinetics are related with blastocyst formation and chromosome

content.

5.

Early compaction is related with good prognosis, while blastocyst collapse, MNB(4), asymmetry(2) and DD is related

with bad prognosis.

6.

The clinical use of time-lapse for embryo selection is able to improve reproductive outcome , taking into account the

algorithm based on exclusion and inclusion criteria, and more stable culture conditions without manipulation .

Concluding remarks

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Thank you for your attention