Comprehensive Chromosome Screening in IVF: Enhancing Outcomes While Reducing the Burden of Care...
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Transcript of Comprehensive Chromosome Screening in IVF: Enhancing Outcomes While Reducing the Burden of Care...
Comprehensive Chromosome Screening in IVF: Enhancing Outcomes While Reducing the Burden of Care
Richard T. Scott, Jr, MD, HCLDClinical and Scientific Director,
Reproductive Medicine Associates of New JerseyProfessor and Director, Reproductive Endocrinology
Robert Wood Johnson Medical School, Rutgers University
aCGH (Lab 1)
SNP array (Lab 3)
qPCR (Lab 2)
46,XY
45,XY,-8
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
John Rock, MD“According to Specifications”
1937Robert Edwards,
PhD“Sexing Rabbit
Blastocysts”
1967
Leeanda Wilton, PhD“Cleavage Bx”
1986
Marilyn MonkMurine Model
PGD Lesch-Nyhan
1987
Yury Verlinsky, PhD
“PB Biopsy”
1988
Audrey Muggleton-Harris, PhD
“Blastocyst Bx”
19881989Elena
Kontogianni, PhD
“PCR of Y in single
blastomeres”
Alan Handyside, PhDSuccessful
Clinical PGD for X Linked Disorders
1990
Eric Forman, MDEnhanced Safety,
Reduced Cost
2013
Santiago Munne, PhD
First FISH PGD
1993
Nathan Treff, PhDValidated 24 Chr .Platform
2008
What do people think about when you say IVF…
Efficiency of Human Reproduction through IVF
0
20
40
60
80
100
%
N=132,874 Mature Follicles
Retrospective Eval.
Proportion leading to delivery:
7% of Follicles
13% of Embryos
21% of 8 Cells
42% of Blastocysts
What might explain the high failure rate?
Estimating implantation potentialBest practices - morphology
X
1 2 3 4 5 6 7
XX
X X XX
Applying Genomics to Clinical EmbryologyIn an ideal world, identifying implantation potential- the “BEST” test would reflect the true state of nature.
0
10
20
30
40
50
60
70
80
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100
Su
stai
ned
Imp
lan
tati
on
Rat
e (%
)
Embryo Number
X
1 2 3 4 5 6 7
X
X
X X X
X
Transfer decisions would be extremely simple
Contemporary Understanding of Maternal Age and Human Embryonic Aneuploidy
22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 450
10
20
30
40
50
60
70
80
90
100
Age (yrs)
Perc
ent
of E
mbr
yos
Whi
ch a
re A
neup
loid
Franasiak et al – Fertil Steril 2014
N=15,169
Is transferring an aneuploid embryo clinically useful?
Gain of 21
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1718 19
2021
22
XChromosome #
Copy
Num
ber
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
One embryo randomized to undergo biopsy
Transfer the embryos
Implantation, Maternal serum sampling for free fetal DNA and Fingerprinting
Cell submitted for eventual aneuploidy screening and fingerprinting
N=113 pairs; 226 embryos
Overall implantation rates
39% reduction insignificant
27% (mean maternal age 32) reported by Gutierrez-Mateo, C., et al. Fertility and sterility 92, 1544-1556 (2009)
In our opinion, day 3 biopsy will soon be of historic interest only
aCGH enhances delivery rates – an RCT
• RCT
• Age– All < 35– Mean age of 31
• Sample Size– 55 aCGH– 48 control
• Significant improvement in outcomes
• Answers one of the four critical validation questionsMonosomy:Trisomy Ratio of 2
Scott et al Fertility and Sterility 2013; 100:697-703
0
10
20
30
40
50
60
70
80
90
100
< 32 33-35 36-38 39-40 41-42
%
Age (yrs)
With greater experience, actual negative
predictive value is ~98.8%
Scott et al Fertil Steril 2012; 97:870-5
22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 > 450
10
20
30
40
50
60
70
80
90
100Chart Title
Age (yrs)
No-
Eupl
oid
Blas
ts R
ate
(%)
The No Transfer Rate with CCS
Franasiak et al – Fertil Steril 2014
N=15,169
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 35 360
5
10
15
20
25
Prop
ortio
n of
Cas
es w
ith th
is
num
ber
of b
last
ocys
ts a
vaila
ble
for
Biop
sy (%
)
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 35 360
10
20
30
40
50
60
70
80
90
100
Cum
ulati
ve P
ropo
rtion
of C
ases
w
ith th
is n
umbe
r of
bla
stoc
ysts
av
aila
ble
for
Biop
sy (%
)
Number of Blastocyts
52% of cases had 3 or fewer evaluable embryos
How Many Embryos Do Patient Undergoing CCS Have?
Franasiak et al – Fertil Steril 2014
N=15,169
Trisomy:Monosomy Ratio by Age
<35 35-37 38-40 41-42 43+0
0.5
1
1.5
2
2.5
3
Age Group (yrs)
Tris
omy:
Mon
osom
y Ra
tio
Franasiak et al – Fertil Steril 2014Key Indicator for QA of your assay
N=15,169 Ratios consistent across nine programs
Clinical ExperienceMisdiagnoses
• 4974 embryos
• 2976 gestations (62.1%)
• 10 errors– 1 tetraploid– 2 monosomies– 7 trisomies
• 3168 transfers
• 2354 ongoing / delivered (72.1%)
• Mean age 38.4 years
• 10 errors– 7 found in losses– 3 found in ongoing preg.
Mosaicism evaluated in 4 samples – 100% mosaic
Clinical Error RatePer embryo 0.2%
Per transfer 0.3%Per ongoing pregnancy 0.1%
Consolidated Pregnancy OutcomesProportion of All Pregnancies
< 35 35 - 38 - 41 - 43+0
10
20
30
40
50
60
70
80
90
CCS - Delivery
No Screening - Delivery
CCS - Clinical Loss
No Screening - Clinical Loss
CCS - Chemical Los
No Screening - Chemical Loss
Maternal Age (yrs)
Clin
ical
Los
s Rat
e (%
)
N=4,754 pregnancies
Scott KL et al – RMA
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
10% 15% 20% 25% 30% 35% 40% 45% 50% 55% 60% 65% 70% 75% 80%0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
2 Blastocyst3 Blastocyst4 Blastocyst
Aneuploidy Rate
Prob
abili
ty o
f at l
east
1 E
uplo
id B
last
ocys
t
<35 35-37 38-40 41-42
21%34%
55%64%
Blastocyst Aneuploidy Rate by Age Group
Forman EJ et al., O-161
Simplified Calculation of Sustained Implantation Rates
Transfer Order:
30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 603032343638404244464850525456586062646668707274767880828486
1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2
Sustained Implantation Rate (%)
Deliv
ery
Rate
(%)
• Start with delivery rate• Draw horizontal line to transfer order• Draw vertical line to attain SIR• Interpolate as needed
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. ASRM Practice Committee, Fertil Steril, 2012. PMID: 221923522. Pinborg A, et al., Acta Obstet Gynecol Scand, 2004. PMID: 154881253. Sazonova A ,et al., Fertil Steril, 2013. PMID: 23219009
The Perception that Patients Love Multiples if They are Born Healthy is not Well Validated
Provided by a patient…
FRESH SET RESULTS IN LOWER DELIVERY RATES THAN DOUBLE EMBRYO TRANSFER (DET)
• 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
Pandian Z et al., Cochrane Database Syst Rev, 2009. PMID: 19370588
SET DET0%
5%
10%
15%
20%
25%
30%
35%
40%
45%
26%30%
13.2%
Livebirth Rate - SET vs. DET
TwinsSingletons
0.5%
The Dropout Rate from IVF is Significant
1 2 3 4 5 6 70%
10%
20%
30%
40%
50%
60%
70%
Dropout/cycleCumm Actual PR
Cycle Number
Perc
ent D
ropo
ut
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
Can1 ≥ 2?
CCS Results in Higher Implantation Rates
Implantation = cardiac activity at time of discharge to obstetrical care (~9 weeks)
Euploid SET Traditional DET0%
10%
20%
30%
40%
50%
60%
70%
80%
66.3%
51.2%
P=0.02
N=83 N=170
Same Delivery Rate:Randomized Controlled Trial
Forman EJ et al. Fertil Steril 2013
0%
10%
20%
30%
40%
50%
60%
70%
80%
61%65%
Delivery Rate Per Patient (n=175)Single euploid blastocyst transfer (N=89)Untested 2-blastocyst transfer (N=86)
P=0.5
P<0.001
Eliminates Multiples
Single euploid blastocyst transfer Untested 2-blastocyst transfer
100%
52%
0%
48%
Singletons Multiples
P<0.001
Forman EJ et al. Fertil Steril 2013
Single euploid blastocyst transfer Grams
Untested 2- blastocyst transfer Grams
1000150020002500300035004000450050005500
Bir
thw
eig
ht
(Gra
ms)
Better Obstetrical Outcomes are Attained CCS/eSET than Conventional Two Embryo Transfer
•Mean Birthweight: 3408 ± 562g – Single euploid 2745 ± 743g – 2-Blastocyst
(P<0.001)
•Low birthweight (<2,500g): 4.4% (2/45) – Single Euploid 31.9% (22/69) – 2-Blastocyst
(P<0.001)
•Very low birthweight (<1,500g): 0% (0/45) – Single Euploid 7.2% (5/69) – 2-Blastocyst (P=0.06)Single
euploid blastocyst transfer
Untested 2-blastocyst
transfer
Better Obstetrical OutcomesReduced Risk of NICU Admission
Untested 2-Embryo Transfer: 479 total days spent in NICU
Euploid eSET: 93 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 PatientsCurrent Standard Of Care
Costs per Delivery*
Singleton $21,458
Twins $104,831
Triplets $407,199
Does not include:Pediatric costs after 28 days of ageDisability costs during bed restLoss 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
CCS / SET National Avg
Regional Avg
$0
$10,000
$20,000
$30,000
$40,000
$50,000
$60,000
$70,000
$80,000Costs per Delivery*• Use actual cost data
• Inclusive of all IVF costs including– IVF cycle costs– CCS costs– Medication costs
• Delivery costs and subsequent hospital stay through 28 days of life
Natural versus Stimulated Folliculogenesis and Embryonic
Aneuploidy• Prospective
observational
• Historic Control
• Ages 21-49 years
• Follicular diameter at retrieval ~ 21 mm
• hCG induced follicular maturation
<30 30+ 35+ 40+ 42+ 45+0
10
20
30
40
50
60
70
80
90
100
Stimulated Natural
Ane
uplo
id (%
)
P=NS
The Natural Cycle Study
Hong ASRM 2014
Patient Acceptance of eSET
CCS Unscreened0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
DETeSET
Franasiak - RMANJ 2013-2015
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 SequencingAlignment
• Aligned relative to a reference genome
Next Generation SequencingDepth of Coverage
• 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
dept
h
Embryo 1 Embryo 3Embryo 2
D1152 D1152
W1282 W1282
D1152
W1282
Cystic Fibrosis Case 1 – Point Mutations
Normal Carrier Affected
Absent Present Present
Absent Absent Present
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.
Next Gen Sequencing for Aneuploidy Screening
Accuracy (n=54)
Per Chromosome: 99.93% (1295/1296)Per Cell: 98.14% (53/54)
Known Mosaic
Time Lapse Observations in the Embryology Laboratory
And others…..
1st 2nd 3rd 4th0
102030405060708090
100ICSI to start of 1st cytokinesis (p=0.61)
Quartile
% E
uplo
id
1st 2nd 3rd 4th0
102030405060708090
100Duration of the 2 cell stage (p=0.88)
Quartile
% E
uplo
id
1st 2nd 3rd 4th0
102030405060708090
100Duration of the 3 cell stage (p=0.12)
Quartile
% E
uplo
id
Time Lapse and AneuploidyTraditional Markers
Hong KH et al – in review
Can Time Lapse Help Distinguish Which Euploid Blasts will Deliver from those Destined to Fail?
Failed
Succesful
1
1.5
2
2.5
3
3.5
4
4.5
5
Transfer Outcome
sy
n_
rm:
tim
e f
rom
sy
ng
am
y t
o 1
st
cy
tok
ine
sis
NO: None of the 5 traditional parameters or 5 additional blast related parameters prognosticate outcome
Temporal data evaluated:
• 5 conventional endpoints through cleavage stage
• Additional temporal endpoints from extended culture:
• First compaction• Morula formation• First cavitation• Blastocyst Expansion• First contraction
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 Unbalanced Unbalanced
Unbalanced Unbalanced Unbalanced Unbalanced Unbalanced
Alternate Segregation Adjacent-1 Segregation Adjacent-2 Segregation
3:1 Segregation 3:1 Segregation 3:1 Segregation 3:1 Segregation 4:0 Segregation
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