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Building robust time-lapse models
Kirstine Kirkegaard
The Fertility Clinic
Aarhus University Hospital
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Time-lapse as a diagnostic test
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Time-lapse
Detailed information
Flexible
Precise*)
Validated?
Morphology
Limited information
Dependent on timing
Subjective
Validated
*Sundvall et al 2013
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Time-lapse incubation
Improved culture?
Improved selection?
Improved treatment?
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Improved pregnancy rates
Assumptions:
1. Culture conditions
• Un-interrupted culture
• Incubator
2. Selection
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Culture conditions
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Embryo selection
Assupmtion: Timing of development can distinguish viable from non-viable embryos
Accuracy and precision
Viable?
What is normal?
Sensitivity and specificity
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Precision and accuracy
ACCURACY:
Dependent on time
between image
recordings
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What is viable?
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Blastocyst development
STUDY ENDPOINT PARAMETERS
Wong 2010 Blastocyst development(n=100)
1st cytokinesis, duration 2-and 3-cell stage
Hashimoto 2012 Blastocyst score (n=80) T(7/8), 3rd cell cycle, duration3-cell stage
Cruz 2012 Blastocyst score (n=834) T(4), duration 3-cell stage, morula, direct cleavage 1-3, uneveness 2-cell stage
Dal Canto 2012 Expanded blastocyst (n=459) T(3)-t(8), duration of all cleavage stages
Hlinka 2012 Blastocyst development(n=180)
Cleavage cycles and interphases.
Conaghan 2013 Blastocyst quality (n=1233) Duration 2- and 3-cell stage
Kirkegaard 2013 High quality blastocysts(n=571)
1st cytokinesis, duration 3-cell stage, direct cleavage 1-3
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AUC: 0.69
Kirkegaard et al, 2013
Prediction of blastocyst
formation vs. pregnancy
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Non-
viable
Viable
What is normal?
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Diagnostic sensitivity and specificity
Depends on the reference intervals/cut off levels!
In practice there is rarely a sharp demarcation between
normal/abnormal
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t5:48–56; s2(t4-t3): ≤0.75; cc2(t3-t2): 5-12
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T3 : 31/34–40 h; cc2 9–12 h; t5: 45-55
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P2 (duration 2 cell stage) 9.33–11.45 hours
P3 (duration 3 cell stage) 0 – 1.73 hours
specificity of 84.7% sensitivity of 38.0%,
PPV of 54.7%, NPV of 73.7%
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usable non-usable
Test usable
TP FP PPV54.7%
Test non-usable
FN TN NPV73.7%
Sensitivitet38.0%
Specificitet84.7%
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Implanted
Not implanted Implantationrate
Usable* n=131 n=445 22,7 %
Non-usable# n=134 n=809 14.2%
Entire cohort n=265 n=1254 17.4%
*P2 : 9.33-11.45 hours and P3: 0-1.73 hours.
#P2 outside 9.33-11.45 hours and P3> 0-1.73 hours.
Kirkegaard et al, 2014
AUC 0.57
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Non-
viable
Viable
What is normal?
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Laboratory/culture conditions
IVF/ICSI
Oxygen
Culture media
Treatment ?
Patients (Age, diagnosis,
BMI, hormones) ?
Cell cycle check points
What is normal?
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25
27,2
39,4 41
52,1
54,5
57,4 59,4
62,5
23,5
26,7
38,2
40,3
50,9
53,7
56,2
59,160,7
20
25
30
35
40
45
50
55
60
65
hrs
aft
er
fert
IVF vs. ICSI
IVF
ICSI
Cruz et al 2013
nIVF =622
nICSI =581
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3,1
14,8 16,8
28,4 31,1
34 36
3,1
14,116,5
27,4
30,533,2
36
0
5
10
15
20
25
30
35
40
hrs
aft
er P
NF
IVF vs ICSI (t0=PNF)
IVF ICSI
Cruz et al 2013
nIVF =622
nICSI =581
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*Students t-test. Blastocyst only nIVF=262 nICSI=375, unpublished data
IVF vs. ICSI
*p<0.001 *p<0.001 *p=0.02
*p=0.001 *p=0.01 *p=0.01
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Wale and Gardner 2010
2-cell 3-cell 4-cell 5-cell 6-cell 7-cell 8-cell EB FB hatching
5% 31,4 51,0 51,9 62,4 63,0 63,4 63,8 89,1 95,8 102,5
20% 31,9 51,7 52,7 65,5 66,1 66,6 67,0 92,6 98,9 105,0
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
110,0
hrs
aft
er
hC
G
5% vs 20 % O2
Culture under 20% oxygen results in cummulative
delayed development and increased developmental
arrest at all stages
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Oxygen-
Human
embryos
20% O2
5%+20% O2
5% O2
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Kirkegaard et al 2013
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25,1
27,4
37,8
40,1
48,8
25,9
29,1
39,541,5
52,2
0
10
20
30
40
50
60
hrs
aft
er
fert
(IC
SI)
single vs sequential media
single
sequential
PNF t3 t5t4t2
nsingle=231 (2 PN=170), nseq=215 (2 PN=149)
71 cycles
**
**
*
Ciray et al 2012
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8,4
23,4
27,3
38,1
40,2
51,2
8,2
23,6
26,7
37,8
40,2
51,1
0
10
20
30
40
50
60
hrs
aft
er
fert
Sage vs Global
Sage
Global
PNF t3 t5t4t22PN
Basile et al 2012
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27,8
38,2
40,3
50,7
53,4
26,9
37,1
39,5
49,8
52,9
0
10
20
30
40
50
60
hrs
aft
er
fert
ilisa
tio
n
stimulation protocol
agonist+hCG
antagonist
t3 t5t4t2 t6
Munoz et al 2012 +
2013
+FSH dose
n=2101
n=713
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Laboratory/culture conditions
IVF/ICSI
Oxygen
Culture media
Treatment ?
Patients (Age, diagnosis,
BMI, hormones) ?
Cell cycle check points
What is normal?
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0
10
20
30
40
50
60
hrs
aft
er f
ert
weight/infertile/fertile
obese infertile
normoweight infertile
donors
t3 t5t4t2
Bellver et al 2013
group age
Obese infertile
34.7 (SD 3.4)
Normo weight infertile
33.3 (SD 2.9)
Donors 27.1 (SD 4.2)
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0
10
20
30
40
50
60
hrs
aft
er f
ert
PCOS
controls
normoandrogenic PCOS
hyperandrogenic PCOS
Wissing et al 2013PNF t3 t5t4t2
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Confounding
Age
timing
pregnancy
aneuploidy
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Logistic regression analysis of the effect of age
and tSB on aneuplodi.
Campbell et al 2014
Age, aneuploidy and tSB
Model effect Estimate Significance of additional effect
Age 0.16 (0.04) 0.016
tSB 0.05 (0.02) 0.0001
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AGE AND BLASTOCYST TIMING
Regression coefficient (95% CI)
P-value Coefficient of determination (R2)
Early blast 0.44 (0.16; 0.71) 0.002 0.02
Full blast 0,42 (0.10;0.74) 0.01 0.02
Clustered linear regression, n=653, npt=149
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AUC: 0.69
Kirkegaard et al, 2013
Prediction of blastocyst
formation vs. pregnancy
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A few words about RCT´s
- Equal distribution of known and unknown confounders
- Elimination of selection bias
Age
timing
pregnancy
aneuploidy
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Laboratory/culture conditions
IVF/ICSI
Oxygen
Culture media
Treatment ?
Patients (Age, diagnosis,
BMI, hormones) ?
Cell cycle check points
What is normal?
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Sensitivity/specificity
Ranking
Confounders
Validation