Will Time Lapse Change Clinical Embryology? - · PDF fileWill Time Lapse Change Clinical Embryology? ... PNf-t2 t0 S2 S3 ... Does size, timing and duration of appearance have

Will Time Lapse Change Clinical

Embryology?

Simon Fishel CARE Fertility Group, UK

COGI

Barcelona – April 2014

CEO – CARE Fertility Group

Preimplantation Genetic Screening (PGS): Practical Options

Disclosure of Conflict of Interest (List)

Shareholder: CARE Fertility Group

Professor Simon Fishel

Time lapse as a study tool

Accurate recording (annotation)

Dysmorphisms and anomalies

Deselection

For each selected anomaly

Overview -1 Cleavage Anomalies

Video Summary info

CARE TLI incidence

Time lapse as a study tool

Accurate recording (annotation)

Morphokinetics

Selection

For each embryo

Overview – 2 Morphokinetic-Based Selection

Video Annotated

MK

CARE Classification

Model

Dynamic v static methodology

More information:

Imaging

Single v 1-300/day

PN

f

t8 t2 t6 t4 t5 t7 t3 tB tSB tM

cc1

ECC3

t8-t4

cc2a cc2b cc3a cc3

d cc3

b

cc3

c

ECC2

t4-t2

ECC1

PNf-t2

t0

S3 S2

Chronological Annotation

CC1 - Cell Cycle 1

CC2a - Cell Cycle single cell division

ECC - Embryo Cell Cycle

t2 - time from t0 to 2 cells

PNf - Pronucleus fading

Consistent annotation is crucial – why?

Pooling of data – intra/inter clinic

Accurate data analysis

Calculation of incidence and impact

Identify of selection and de-selection criteria

Set key performance indicators

Monitor changes in practice

Develop in house embryo selection models

It allows

Intr

ac

yto

pla

smic

• Granulation

• Refractile bodies

• Vacuolation

• sERC

• MN

• Pronuclei

extr

ac

yto

pla

smic

• Polar body

• PV space

• Zona Pellucida

• Fragments

mo

rph

okin

etic

• Time to cell stages

• Durations

• Dynamics e.g

• Pronuclei

•Compaction

• Blastulation

• Strings

• Reverse cleavage

Nothing can hide from us now!

A brief look at 6 Dysmorphisms / Anomalies

1. sER clustering

2. Vacuolation

3. PN alteration

5. Blastocoelic

Strings

6. Reverse

cleavage

1. Smooth endoplasmic reticulum clusters

(sERC)

ER = source and regulator

of Ca2+ signals

Vital at

activation/fertilisation

BUT sER aggregates -

‘Pronucleus sized

translucent vacoules’

Associated with oocyte

ageing

Represent degenerative

processes

Development to

blastocyst can occur

At least 1 Beckwith‐Wiedemann

Syndrome reported

Alpha/ESHRE

Consensus:

‘Do not

transfer’

Ebner 2008

6% of patients 1.8% oocytes

Related to stimulation

dosage and duration

Significantly reduced

fertilisation and blastulation

rates

Trend for early delivery and

reduced birth weight

Miwa 2013 ESHRE

7.3% of cycles affected

Can we prevent sERC?

‘in most patients’

Earlier hCG

Modified stimulation

protocol

How common is sERC? - published facts and figures, static observation

‘the worst (oocyte)

dysmorphism observable’ (Ebner, 2011

• About 1 in 300 inseminated eggs

exhibited sERC

• Fertilisation ~40%

2. Vacuolation

Small <10um ‘unlikely

to be of biological

significance’

Large>14µm vacuoles

associated with

fertilisation failure.

Can result in lower

blastulation

Poorly understood

New video required

Does size, timing

and duration of

appearance have

significance?

Vacuoles, CARE data – 7552 x 2PN

SOP - Observe and

record their presence.

Tendency to avoid

More study required

CARE incidence

1.2% embryos

First appearance

0.35-81.4hpi

Of 23 ET’d 9 x CP (39%)

3. Is a 2PN always a 2PN?!

Standard method of

confirming fertilisation-

2PN

17+/-1 hour hpi

With time lapse - How

often would a

decision at 18hpi

change?

3PN → 2PN

3. Is a 2PN always a 2PN?!

1PN → 2PN

PN ‘alteration’ CARE data – 7552 x 2PN

1/100 ‘2PNs’ may not

be all that they seem!

TLI allows identification

& exclusion of these

1.1%

82 x 2PN

Became 1PN, 3PN or 4PN

0.2%

13 x 2PN

1PN

0.9%

67x 2PN 3PN

0.03%

2 x 2PN 4PN

Can we/should we fine

tune PN check for

standard practice?

t8 t2 t6 t4 t5 t7 t3 tB tS

B

tM

cc2a

cc2b

ECC2

t4-t2

t0

<5h

• ‘P2’ – Wong et al 2010 - 11.1± 2.2h • CC2 – Meseguer et al 2012 - </=11.9h

• Interphase 2 – Hlinka et al 2012 - 11±1h

• DC2-3 - Rubio et al 2012 - <5h

An important morphokinetic marker

ECC2 (Embryo Cell Cycle 2)

4. Direct or rapid division 13 cells

Novel event first

described 2011

(Rubio et al)

‘Rapid cleavage 2 to

3 cells in less

than 5 hours’

May be caused by

spindle or

cell cycle errors

Insufficient time for

DNA replication?

4.‘Direct cleavage’ 1-3

Incidence 26.3%

CARE CC2 data – <5h very common

IPR 1.2%

and b. Rapid 13

(<5 hours at 2 cells) a. ‘Direct’ 13

no time at 2 cells

We considered ‘direct or rapid’ cleavage

to 3 cells

t3-t2

Incidence of embryos

exhibiting

Direct or rapid13

<5h = 27.9%

<2h = 19.8%

CARE recent data

supports de-selection

where Direct or

rapid13 <2h

Direct or rapid 13

<2h

2.9% (FH)*

1.6% (LB)

P<0.0001 High

incidence!

This is very common! CARE data – 7552 x 2PN embryos

5. Blastocoelic Strings

Commonly observed traversing the blastocoel

Persistence in the exp. blastocyst associated

with poor embryo quality, poor culture

conditions or breakdown in polarisation (Scott

2000;Hardarson 2012).

Recent time lapse work – incidence 62%

(n=113)

Significantly higher in implanted v non

implanted blastocysts (Cater et al, 2014)

6. Reverse cleavage/cell merging

Static study (Balakier 2000)

cryopreserved embryos – 4.6%

day 2; 1.5% day 3.

2012 time-lapse study

reported 6.8% incidence

(Hickman).

Suggested link between RC

and MN.

Lower rate in antagonists

cycles compared with

agonist.

Blastocyst development was

not impaired.

CARE current data - 4.8%

(129/2714) transferred

embryos had undergone RC

15.7% KID positive

Live birth reported (Stecher

2014)

Time lapse brings new

opportunities

With strict annotation Develop understanding

More precise

identification of

anomalies

Relate morphokinetics to

outcome

Improve embryo

selection

Follow on TLI studies

sERC & vacuoles – size

timing, duration of

appearance, impact on

embryo development and

outcome.

PN dynamics

Active use of ‘direct

cleavage’ in selection models and hunt for more

using known outcome

data.

Summary of Anomalies

Morphokinetics & Ploidy

98 Blastocysts: Annotated & Blind to Ploidy,

tPNfaded Time for PN fading (hours)

t2 Time from insemination to divisions to two cells complete

(hours)

t3 Time from insemination to divisions to three cells

complete (hours)

t5 Time from insemination to divisions to five cells complete

(hours)

t8 Time from insemination to divisions to eight cells

complete (hours)

tSC Time from insemination to start of compaction (hours)

tM Time from insemination to morula (hours)

tSB Time from insemination to start of blastulation (hours)

tB Time from insemination to Blastocyst formation complete

(hours)

tEB Time from insemination to expanded blastocyst (hours)

tHB Time from insemination to hatched blastocyst (hours)

cc2 The time period of the second cell cycle (t3-t2)

cc3 The time period of the third cell cycle (t5-t3)

s2 The time period of the synchrony of the first cell cycle (t4-

t3)

s3 The time period of the synchrony of the first cell cycle (t8-

t5)

Blastulation The time period of blastulation (tB-tSB)

MN2 Multinuclearity at the two cell stage (TRUE/FALSE)

MN4 Multinuclearity at the four cell stage (TRUE/FALSE)

1->3 Direct cleavage from one to three cells

2->5 Direct cleavage from two to five cells

Two Candidates For Aneuploidy Risk Classification

Model

tSB & tB

tSB tB

The model - classifying aneuploidy risk by

timing alone

Partition embryos

according to tSB and tB

Aneuploidy incidence

per risk class

69% overall

36% in low risk

61% in medium risk

100% in high risk

122.9h

96.2h

Use MK Data When Embryo Fate -

implantation or live birth outcome - Is Known

And compare rates of implantation or

live birth by MK variables (KID rate)

positive

KID negative negative Positive

No KID**

DET

Pregnancy

loss 2 x implantations

lost

1 x

implantations

lost

One Live Birth

KID positive

Two Live Birth

KID positive No Live Birth

KID negative

No LB KID

negative

2 x LB KID

negative

SET

Retrospective Analysis of Embryos in Standard

Treatment Cycles Without PGS – LIVE BIRTH

KID (LB)

tB, hours post insemination

80 90 100 110 120 130 140

tSB

, ho

urs

po

st in

se

min

atio

n

80

90

100

110

120

130

140

Negative

Positive

KID (LB)

Time of start of Expansion, hours post insemination

80 90 100 110 120 130 140

Tim

e o

f sta

rt o

f B

lastu

latio

n, ho

urs

po

st in

se

min

atio

n

80

90

100

110

120

130

140

Negative

Positive

61%

LB

16% LB

0% LB

Retrospective Analysis of Embryos in Standard

Treatment Cycles Without PGS – LIVE BIRTH

Independent Model Validation 27 clinics worldwide

70

80

90

100

110

120

130

140

80 90 100 110 120 130 140 150

tSB

(h)

tB (h)

Modelling embryo implantation (n = 536)

Not implanted

Implanted (GS/FHB)Implantation rate = 0.65

Implantation rate = 0.37 Implantation rate = 0.10

unpublished

Low risk 65%

Medium risk

37%

High risk

10%

Effect of Age

Demonstrates that the levels of

aneuploidies may vary greatly in

different age groups, underlining

that the aneuploidy risk

classification model should not

be used to determine the actual

risk of aneuploidy (e.g. 65.6%

chance of aneuploidies) but to

assess the relative risk of

aneuploidies (e.g. embryo x has

a greater risk of being aneuploid

compared to embryo y)

Morphokinetics v Age

Black line is the trend (linear

regression) line, the

Blue lines indicate the 95%

confidence interval

Green lines indicate the 95%

prediction interval.

n=1517

n=1271

Age is a weak correlation

Controlling factor appears to be

Aneuploidy

Aneuploidy / Morphokinetics: Basile et al 2014

• n=504

• ‘Chromosomally normal

and abnormal embryos have different kinetic

behaviour’

• MK biomarkers of ploidy t5-

t2 and cc3 (t5-t3)

Time lapse studies linking MKs and ploidy

• Prediction of Ploidy and implantation using a risk model

– Campbell et al 2013 a, b RBM Online

• Prediction of Aneuploidy related to morphokinetic classification

– Basile et al ASRM13:O-21(linear regression model); Basile et al 2014 F&S

• Direct cleavage and impact on aneuploidy

– Zaninovic et al. (ASRM13-P-327)

• Early morphokinetic parameters important for ploidy prediction

– Basile et al. (ESHRE13-O-101)

• Compaction and Ploidy

– Melzer et al. (ASRM13-P-213); Montgomery et al. (ESHRE13-O-003)

• Aneuploidy prediction from late morphokinetic parameters

– Nagorny et al. (ASRM13-P-211)

• Multinucleation and ploidy

– Melzer et al (ASRM 13-P-213); Mazur et al.

(ASRM13-O-223)

Next steps?

Consensus

Best practice

Collaboration

Amassing data

Improving outcomes

Next steps

Next steps?

It already

has!

Will Time Lapse Change Clinical Embryology?

Thank you for listening

and to all the CARE Fertility team