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![Page 1: 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](https://reader031.vdocument.in/reader031/viewer/2022022503/5aaf38ca7f8b9adb688d5ee4/html5/thumbnails/1.jpg)
Will Time Lapse Change Clinical
Embryology?
Simon Fishel CARE Fertility Group, UK
COGI
Barcelona – April 2014
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CEO – CARE Fertility Group
Preimplantation Genetic Screening (PGS): Practical Options
Disclosure of Conflict of Interest (List)
Shareholder: CARE Fertility Group
Professor Simon Fishel
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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
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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
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Dynamic v static methodology
More information:
Imaging
Single v 1-300/day
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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
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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
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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!
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A brief look at 6 Dysmorphisms / Anomalies
1. sER clustering
2. Vacuolation
3. PN alteration
5. Blastocoelic
Strings
6. Reverse
cleavage
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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’
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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%
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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
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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%)
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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
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3. Is a 2PN always a 2PN?!
1PN → 2PN
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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?
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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)
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4. Direct or rapid division 13 cells
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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
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Incidence 26.3%
CARE CC2 data – <5h very common
IPR 1.2%
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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
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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
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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)
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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)
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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
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Morphokinetics & Ploidy
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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
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Two Candidates For Aneuploidy Risk Classification
Model
tSB & tB
tSB tB
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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
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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
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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
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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
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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%
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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)
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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
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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)
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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)
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Next steps?
Consensus
Best practice
Collaboration
Amassing data
Improving outcomes
Next steps
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Next steps?
It already
has!
Will Time Lapse Change Clinical Embryology?
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Thank you for listening
and to all the CARE Fertility team