Comprehensive Chromosome Screening in IVF:
Enhancing Outcomes While
Reducing the Burden of Care
aCGH (Lab 1)
45,XY,-8
qPCR (Lab 2)
SNP array (Lab 3)
46,XY
Richard T. Scott, Jr, MD, HCLD
Clinical and Scientific Director,
Reproductive Medicine Associates of New Jersey
Professor and Director, Reproductive Endocrinology
Robert Wood Johnson Medical School, Rutgers University
Disclosures
• Board Member – FEC
• No compensation of any kind
• No financial interest in any test or diagnostic
platform
The Road to Clinically Effective Preimplantation Genetics
Yury Verlinsky,
PhD
“PB Biopsy”
1986
Leeanda Wilton, PhD
“Cleavage Bx”
1987
1967
1937
John Rock, MD
“According to
Specifications”
Santiago
Munne, PhD
First FISH PGD
2008
Nathan Treff, PhD
Validated 24 Chr
.Platform
Marilyn Monk
Murine Model
PGD Lesch-Nyhan
Robert Edwards,
PhD
“Sexing Rabbit
Blastocysts”
1993
1988
1990
Alan Handyside, PhD
Successful
Clinical PGD for X
Linked Disorders
2013
Eric Forman, MD
Enhanced Safety,
Reduced Cost
1989
1988
Elena
Kontogianni,
PhD
“PCR of Y in
single
blastomeres”
Audrey
MuggletonHarris, PhD
“Blastocyst Bx”
What do people think about when you say IVF…
Efficiency of Human Reproduction through IVF
100
Retrospective Eval.
80
Proportion leading
to delivery:
60
7% of Follicles
40
13% of Embryos
%
20
0
N=132,874 Mature Follicles
21% of 8 Cells
42% of Blastocysts
What might explain the high failure rate?
Estimating implantation potential
Best practices - morphology
X
1
X
X
2
3
X
X
X
4
5
6
X
7
Applying Genomics to Clinical Embryology
Sustained Implantation Rate (%)
In an ideal world, identifying implantation potentialthe “BEST” test would reflect the true state of nature.
100
90
80
70
60
50
40
30
20
10
0
X
X
X
X
1
2
X
3
X
X
4
5
Embryo Number
6
Transfer decisions would be extremely simple
7
Contemporary Understanding of Maternal Age and
Human Embryonic Aneuploidy
100
N=15,169
Percent of Embryos Which are Aneuploid
90
80
70
60
50
40
30
20
10
0
22
23
24
25
26
27
28
29
30
31
32
33
34
Age (yrs)
Franasiak et al – Fertil Steril 2014
35
36
37
38
39
40
41
42
43
44
45
Copy Number
Is transferring an aneuploid embryo
clinically useful?
Gain of 21
1
2
3
4
5
6
7
8
9
Chromosome #
10
11
12
13 14 15 16 17
18 19
21
20 22
X
What are the “Burdens” of CCS
Thus the real questions are:
1. Safely attaining
embryonic DNA
2. Predictive values of the
techniques
3. Proportion of euploid
embryos that will fail
4. Cost effectiveness
Does Embryo Biopsy Impact the
Developmental Potential of the Oocyte
Routine IVF
Care through Retrieval
Identify mature oocytes
ICSI, culture, and select 2
best embryos for transfer
Transfer the
embryos
Cell submitted for eventual
aneuploidy screening and
fingerprinting
Implantation, Maternal serum sampling
for free fetal DNA and Fingerprinting
One embryo
randomized to
undergo biopsy
N=113 pairs; 226 embryos
Overall implantation rates
39% reduction
insignificant
In our opinion, day 3
biopsy will soon be of
historic interest only
27% (mean maternal age 32) reported by Gutierrez-Mateo, C., et al. Fertility and sterility 92, 1544-1556 (2009)
aCGH enhances delivery
rates – an RCT
• RCT
• Age
– All < 35
– Mean age of 31
• Sample Size
– 55 aCGH
– 48 control
• Significant improvement
in outcomes
Monosomy:Trisomy Ratio of 2
• Answers one of the four
critical validation
questions
Scott et al Fertility and Sterility 2013; 100:697-703
100
90
80
70
60
%
50
40
With greater experience,
actual negative
predictive value is
~98.8%
Scott et al Fertil Steril 2012; 97:870-5
30
20
10
0
< 32 33-35 36-38 39-40 41-42
Age (yrs)
The No Transfer Rate with CCS
100
90
No-Euploid Blasts Rate (%)
80
N=15,169
70
60
50
40
30
20
10
0
22
23
24
25
26
27
28
29
30
31
32
33
34
Age (yrs)
Franasiak et al – Fertil Steril 2014
35
36
37
38
39
40
41
42
43
44
> 45
How Many Embryos Do Patient Undergoing CCS Have?
Cumulative Proportion of Cases
with this number of blastocysts
available for Biopsy (%)
100
90
80
70
60
52% of cases had 3 or fewer evaluable embryos
50
40
30
20
10
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
Proportion of Cases with this
number of blastocysts available for
Biopsy (%)
25
N=15,169
20
15
10
5
0
1
2
3
4
5
6
7
Franasiak et al – Fertil Steril 2014
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
Number of Blastocyts
Trisomy:Monosomy Ratio by Age
Ratios consistent across nine programs
N=15,169
Trisomy:Monosomy Ratio
3
2.5
2
1.5
1
0.5
0
<35
35-37
38-40
Age Group (yrs)
41-42
43+
Key Indicator for QA of your assay
Franasiak et al – Fertil Steril 2014
Clinical Experience
Misdiagnoses
Clinical Error Rate
Per embryo
Per transfer
Per ongoing pregnancy
0.2%
0.3%
0.1%
• 4974 embryos
• 3168 transfers
• 2976 gestations (62.1%)
• 2354 ongoing / delivered
(72.1%)
• 10 errors
– 1 tetraploid
– 2 monosomies
– 7 trisomies
• Mean age 38.4 years
• 10 errors
– 7 found in losses
– 3 found in ongoing preg.
Mosaicism evaluated in 4 samples – 100% mosaic
Consolidated Pregnancy Outcomes
Proportion of All Pregnancies
90
80
N=4,754 pregnancies
Clinical Loss Rate (%)
70
60
CCS - Delivery
50
No Screening - Delivery
40
CCS - Clinical Loss
No Screening - Clinical Loss
30
CCS - Chemical Los
20
No Screening - Chemical Loss
10
0
< 35
Scott KL et al – RMA
35 -
38 41 Maternal Age (yrs)
43+
Kulkarni D et al, New Engl J Med, 2014. PMID: 24304051
Kulkarni D et al, New Engl J Med, 2014. PMID: 24304051
With >2 blastocysts, even patients at high aneuploidy
risk are very likely to have a euploid blastocyst
Probability of at least 1 Euploid Blastocyst
100%
90%
80%
70%
60%
50%
Blastocyst Aneuploidy
Rate by Age Group
40%
30%
55%
21%
64%
34%
20%
10%
0%
<35
35-37
38-40
41-42
Forman EJ et al., O-161
10% 15% 20% 25% 30% 35% 40% 45% 50% 55% 60% 65% 70% 75% 80%
Aneuploidy Rate
2 Blastocyst
3 Blastocyst
4 Blastocyst
Delivery Rate (%)
Simplified Calculation of
Sustained Implantation Rates
86
84
82
80
78
76
74
72
70
68
66
64
62
60
58
56
54
52
50
48
46
44
42
40
38
36
34
32
30
•
•
•
•
30
Start with delivery rate
Draw horizontal line to transfer order
Draw vertical line to attain SIR
Interpolate as needed
32
34
36
38
40
42
44
46
48
50
52
54
56
Sustained Implantation Rate (%)
Transfer Order:
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2
58
60
Singleton Term Delivery: The Ideal IVF Outcome
• IVF twin pregnancies are at an increased risk of:
– Preeclampsia (2-fold risk increase)1
– Extreme prematurity (7.4-fold increase delivery
<32 wks) 2
– NICU admission (3.8-fold increased risk)2
– Perinatal Death (2-fold increase)2
• Two IVF singleton deliveries have better obstetrical
outcomes than one IVF twin delivery3
1.
2.
3.
ASRM Practice Committee, Fertil Steril, 2012. PMID: 22192352
Pinborg A, et al., Acta Obstet Gynecol Scand, 2004. PMID: 15488125
Sazonova A ,et al., Fertil Steril, 2013. PMID: 23219009
Provided by a patient…
FRESH SET RESULTS IN LOWER DELIVERY RATES THAN
DOUBLE EMBRYO TRANSFER (DET)
Livebirth Rate - SET vs. DET
45%
40%
13.2%
35%
30%
25%
0.5%
Twins
20%
15%
Singletons
26%
30%
10%
5%
0%
SET
• Cochrane Review of 6
randomized trials from
1999-2006 (N = 1,257)
• Young, good prognosis
patients with “top
quality” embryos
available
• Slightly more singletons
after DET
DET
Pandian Z et al., Cochrane Database Syst Rev, 2009. PMID: 19370588
The Dropout Rate from IVF is Significant
70%
60%
Percent Dropout
50%
40%
Dropout/cycle
30%
Cumm Actual PR
20%
10%
0%
1
2
3
4
5
Cycle Number
Source: Schroder AK: Cumulative pregnancy rates and drop out rates of a German IVF
programme: 4, 102 cycles in 2,130 patients. RBM Online (2004) 8:600-606
6
7
1
Can
≥
2?
CCS Results in Higher Implantation Rates
80%
70%
P=0.02
66.3%
60%
51.2%
50%
40%
N=83
N=170
Euploid SET
Traditional DET
30%
20%
10%
0%
Implantation = cardiac activity at time of discharge to obstetrical care (~9 weeks)
Same Delivery Rate:
Randomized Controlled Trial
80%
70%
60%
Delivery Rate Per Patient (n=175)
Single euploid blastocyst transfer (N=89)
Untested 2-blastocyst transfer (N=86)
P=0.5
61%
65%
50%
40%
30%
20%
10%
0%
Forman EJ et al. Fertil Steril 2013
Eliminates Multiples
Singletons
100%
Multiples
P<0.001
P<0.001
52%
48%
0%
Single euploid blastocyst transfer
Untested 2-blastocyst transfer
Forman EJ et al. Fertil Steril 2013
Better Obstetrical Outcomes are Attained CCS/eSET
than Conventional Two Embryo Transfer
5500
•Mean Birthweight:
5000
3408 ± 562g – Single euploid
2745 ± 743g – 2-Blastocyst
(P<0.001)
Birthweight (Grams)
4500
4000
•Low birthweight (<2,500g):
3500
4.4% (2/45) – Single Euploid
31.9% (22/69) – 2-Blastocyst
(P<0.001)
3000
2500
2000
•Very low birthweight (<1,500g):
1500
1000
Single
euploid
Untested 2Single
Untested
blastocyst
blastocyst
euploid
2-blastocyst
transfer
Grams transfer
Grams
blastocyst
transfer
transfer
0% (0/45) – Single Euploid
7.2% (5/69) – 2-Blastocyst (P=0.06)
Better Obstetrical Outcomes
Reduced Risk of NICU Admission
Euploid eSET:
93 total days spent in NICU
Untested 2-Embryo Transfer:
479 total days spent in NICU
Forman EJ et al, Am J Obstet Gynecol, 2014. PMID: 24145186
Fewer “Big Ticket” Hospital Admissions
• Mean total hospital and
IVF charges per delivery:
– $73,407 (euploid eSET)
– $111,488 (untested 2-ET)
• ~33% reduction in costs
per delivery
• A $38,000 difference
per IVF delivery would
represent $1.9 B savings
on health care costs
with ~50,000 IVF
deliveries annually
Forman EJ et al
Obstetrical Costs for 100 Patients
Current Standard Of Care
Costs per Delivery*
Singleton
$21,458
Twins
$104,831
Triplets
$407,199
Does not include:
Pediatric costs after 28 days of age
Disability costs during bed rest
Loss of productivity in the work place
Lemos et al Am J Obstet Gynecol 2013; 209:586
Overall Cost to Provide Care
CCS with SET versus Conventional Treatment
• Use actual cost data
– IVF cycle costs
– CCS costs
– Medication costs
Thousands
• Inclusive of all IVF costs
including
Costs per Delivery*
$80
$70
$60
$50
$40
$30
$20
• Delivery costs and
subsequent hospital stay
through 28 days of life
$10
$0
CCS / SET National Regional
Avg
Avg
Natural versus Stimulated
Folliculogenesis and Embryonic
Aneuploidy
The Natural Cycle Study
100
P=NS
90
Aneuploid (%)
80
70
60
50
40
30
20
10
0
<30
Hong ASRM 2014
30+
35+
Stimulated
40+
42+
Natural
45+
Patient Acceptance of eSET
100%
90%
80%
70%
60%
50%
DET
40%
eSET
30%
20%
10%
0%
CCS
Franasiak - RMANJ 2013-2015
Unscreened
Follow Up on Prospective Trials
The Future
How NGS Works
• Amplification of limited starting material
• Millions of small fragments are generated…
Embryo
Biopsy
Next Generation Sequencing
Alignment
• Aligned relative to a reference genome
Next Generation Sequencing
Depth of Coverage
depth
• Assemble all the fragments
• The number of fragments which “fit” into a given area of the
genome defines the DEPTH
• High number of fragments allows each base pair to be
sequenced/ evaluated thousands of times in a single assay
Cystic Fibrosis Case 1 – Point Mutations
Normal
Carrier
Affected
Embryo 1
Embryo 2
Embryo 3
D1152
D1152
D1152
Absent
W1282
Absent
Present
Present
W1282
W1282
Absent
Present
Next Gen Sequencing for Aneuploidy Screening
Accuracy (n=54)
Per Chromosome: 99.93% (1295/1296)
Per Cell:
98.14% (53/54)
Known
Mosaic
Universal Primer with new UP reference (SNPs replaced with Ns), on 54 samples.
Some small improvements.
Coverage per sample is on next page. Known aneuploidies in red; known XX in cyan.
Time Lapse Observations in the
Embryology Laboratory
And others…..
% Euploid
100
90
80
70
60
50
40
30
20
10
0
ICSI to start of 1st cytokinesis (p=0.61)
Time Lapse and Aneuploidy
Traditional Markers
1st
2nd
3rd
4th
Quartile
% Euploid
% Euploid
Duration of the 2 cell stage (p=0.88)
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
Duration of the 3 cell stage (p=0.12)
1st
2nd
3rd
4th
Quartile
1st
2nd
3rd
Quartile
4th
Hong KH et al – in review
Can Time Lapse Help Distinguish Which Euploid
Blasts will Deliver from those Destined to Fail?
Temporal data evaluated:
syn_rm: time from syngamy to 1st cytokinesis
5
• 5 conventional endpoints
through cleavage stage
4.5
4
3.5
3
2.5
2
1.5
1
Failed
Succesful
• Additional temporal
endpoints from extended
culture:
• First compaction
• Morula formation
• First cavitation
• Blastocyst Expansion
• First contraction
Transfer Outcome
NO:
None of the 5 traditional parameters or 5 additional blast
related parameters prognosticate outcome
Duplications and Deletions
• Arrays versus metaphase spread at time of amniocentesis
• Wopner et al NEJM December 2012
• Population mostly advanced maternal age
• Duplications and Deletions
– Evaluated 80 dup/del’s with known clinical phenotype
– 1 in 72 fetus’s had a clinically significant dup/del!
– Risk considerably higher than for ongoing aneuploid gestation
• Possible to simultaneously screen for these dup/del’s and
aneuploidy with multiple platforms
• Extensive validation required
Some Disagree
with PGS
•
•
•
•
•
•
•
•
All embryo selection techniques are detrimental
Inappropriate to use “Implantation Rates” as an endpoint
“it can be questioned whether all patients will ever be able to understand all of the complexities
concerning PGS”
“cost-effectiveness is being forgotten”
“In our view evaluating the (cost-)effectiveness of medical treatments is by far the greatest
challenge in current day medicine, especially in an era where health care costs continue to
increase to the extent where they are the number one item of expense for many governments
across the globe”
“evidence is now accumulating that all embryos in an IVF cycle can be cryopreserved and
transferred in subsequent cycles without impairing, and maybe even improving, the cumulative
pregnancy rate of that IVF cycle”
Embryo selection should therefore not be used to select out embryos, but only to determine the
order in which the embryos will be transferred, as the time to pregnancy can be improved by
embryo selection, if embryos with the highest implantation potential are transferred first.
Culturing to the blastocyst may be harmful
Chromosome Pairing Before Segregation
is Essential to Allow Crossing Over –
Even with Balanced Translocations
Normal
Translocation Carrier
Normal Chromosome Segregation
Normal
Translocations - Chromosome Segregation
Alternate
Segregation
Translocations - Chromosome Segregation
Adjacent-1
Segregation
Translocations - Chromosome Segregation
Adjacent-2
Segregation
Normal
Normal Balanced
Alternate Segregation
Unbalanced
Unbalanced
Adjacent-1 Segregation
Adjacent-2 Segregation
Unbalanced
Unbalanced
Unbalanced
Unbalanced
Unbalanced
3:1 Segregation
3:1 Segregation
3:1 Segregation
3:1 Segregation
4:0 Segregation
The RMA New Jersey Research Team
Clinical Embryology
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Xinying Li
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