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
aCGH (Lab 1)
SNP array (Lab 3)
qPCR (Lab 2)
46,XY
45,XY,-8
Disclosures
• Member of the Board of Directors of a Not-For-Profit which provides embryonic aneuploidy screening
• NO compensation of any kind for service as a board member
• No compensation of any kind from any aspect of the evaluation of embryo biopsies
• Not-For-Profit (Foundation for Embryonic Competence; FEC) uses funds to sponsor research
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?
Is the interpretation of PGD objective? Hybridization for 13, 18, 21, X and Y
FISH for Aneuploidy Screening
Novel Comprehensive Chromosome Screening Technologies
aCGH (Lab 1)
SNP array (Lab 3)
qPCR (Lab 2)
46,XY
45,XY,-8
NextGen
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 questions Monosomy:Trisomy Ratio of 2
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, the actual negative predictive value is
~98.8%
Scott et al Fertil Steril 2012; 97:870-5
~4% false abnl rate
Clinical Experience Misdiagnoses
• 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 Rate Per embryo 0.2%
Per transfer 0.3% Per ongoing pregnancy 0.1%
Werner et al – Fertil Steril ePub 2014
0
1
2
3
4
5
6
7
8
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X/Y
Pro
bab
ility
Of
Ch
rom
oso
me
Bei
ng
An
eup
loid
Per
Bio
psy
Chromosome
Aneuploidy Rate by Chromosome
N=15,169
Franasiak et al – in review
Capture Rate • 5 Chromosome FISH – 21.8% • 9 Chromosome FISH – 59.8%
Contemporary Understanding of Maternal Age and Human Embryonic Aneuploidy
0
10
20
30
40
50
60
70
80
90
100
22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
Per
cen
t o
f Em
bry
os
Wh
ich
are
An
eup
loid
Age (yrs)
Franasiak et al – Fertil Steril 2014
N=15,169
0
10
20
30
40
50
60
70
80
90
100
<35 36-38 39-40 41-42 >42
Maternal Age (yrs)
Day 3 Bx
Blastocyst Bx
Harton GL et al Fertil Steril 2013 100:1695-1703
%
Sample Size Day 3: 3,412 Blast: 2,467
0
10
20
30
40
50
60
70
80
90
100
22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 > 45
No
-Eu
plo
id B
last
s R
ate
(%
)
Age (yrs)
The No Transfer Rate with CCS
Franasiak et al – Fertil Steril 2014
N=15,169
0
5
10
15
20
25
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
Pro
po
rtio
n o
f C
ases
wit
h t
his
n
um
ber
of
bla
sto
cyst
s av
aila
ble
fo
r B
iop
sy (
%)
0
10
20
30
40
50
60
70
80
90
100
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
Cu
mu
lati
ve P
rop
ort
ion
of
Cas
es
wit
h t
his
nu
mb
er o
f b
last
ocy
sts
avai
lab
le f
or
Bio
psy
(%
)
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
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45+
Nu
mb
er o
f B
last
ocy
sts
Bio
psi
ed (
n)
Maternal Age (yrs)
Mean Median Minimum Maximum
Number of Embryos Evaluated per Cycle Relative to Maternal Age
Franasiak et al 2014
Trisomy:Monosomy Ratio by Age
0
0.5
1
1.5
2
2.5
3
<35 35-37 38-40 41-42 43+
Tris
om
y:M
on
oso
my
Rat
io
Age Group (yrs)
Franasiak et al – Fertil Steril 2014 Key Indicator for QA of your assay
N=15,169 Ratios consistent across nine programs
0
2
4
6
8
10
12
14
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X/Y
Re
lati
ve I
ncr
eas
e in
An
eu
plo
idy
Rat
e R
ela
tive
to
Age
Gro
up
wit
h L
ow
est
Ris
k (A
ge
26
-30
)
Chromosome
>26 26-30 31-34 35-37 38-40 41-42 43+Franasiak 2014
Changes in Aneuploidy Rates by Chromosome with Increasing Maternal Age
0
2
4
6
8
10
12
14
< 35 35-37 38-40 41-42 43+
Me
an E
rro
r R
ate
pe
r C
hro
mo
som
e in
Eac
h S
tru
ctu
ral C
lass
acrocentric metacentric submetacentric
Franasiak 2014
Chromosome Structure, Embryonic Aneuploidy, and Maternal Age
Acrocentric: 13, 14, 15, 21, 22 Metacentric: 1, 3, 16, 19, 20
All fifteen possibilities have been observed.
Non-disjunction
Premature Separation of Sister
Chromatids
Normal
CHARACTERIZATION OF THE SOURCE OF HUMAN EMBRYONIC ANEUPLOIDY The Sequential Bx Study
• Population – 1104 aneuploid chromosomes
– 786 embryos
– 198 patients
• Microarray PGD on – 1st polar body
– 2nd polar body
– Blastomere or Trophectoderm
• Fingerprinting done on aneuploidy chromosomes at embryonic level to determine if mitotic or paternal
Northrop et al and Treff et al 2010
PSSC is the principal maternal error in meiosis I
23
89.5%
10.5%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
MI PSSC MI ND
Pe
rce
nt
of
An
eu
plo
idy
Origins of MI Aneuploidy
p<0.01 This provides an opportunity for reciprocal errors
Developmental Stage of Error Versus Maternal Age
0
5
10
15
20
25
30
35
40
45
50
< 35 35-37 38-40 41-42
Pro
po
rtio
n o
f Er
rors
(%
)
Maternal Age (yrs)
MI error
MII error
N=327
P=NS
Uniparental Isodisomy – Does self correction exist? Characterization of single cells from established cell lines
0
10
20
30
40
50
60
70
80
<35 36-38 39-40 41-42 >42
Maternal Age (yrs)
Day 3 Bx
Blastocyst Bx
Harton GL et al Fertil Steril 2013 100:1695-1703
Imp
lan
tati
on
Rat
e %
Sample Size Day 3: 508 Blast: 543
0
10
20
30
40
50
60
70
80
<25 26-29 30-34 35-37 38-40 41-42 43+
Sust
ain
ed
Imp
lan
tati
on
Rat
e (
%)
Maternal Age (yrs)
N=18,567
Why do 30-40% of high-quality embryos still fail to implant?
• Embryonic factors
– Non-chromosomal
• indels, methylation
– Mosaicism
– Mitochondrial heteroplasmy
• Uterine factors
– Endometrial receptivity
• Timing
• Infection/Inflammation, Microbiome
• Endometrial contractility
Mosaicism in Human Embryos
• Only evaluable in arrested embryos (upward bias)
• Cleavage stage
– Embryo biopsied in its entirety into individual cells
– Each cell analyzed
• Blastocyst – Three widely separated
biopsies from the trophectoderm
– One biopsy from the inner cell mass
• N=614 embryos
0
5
10
15
20
25
30
35
40
45
50
Day 3 Blastocyst
Em
bry
os
wit
h a
ny
evid
ence
of
Mo
saic
ism
(%
)
P=NS
Outcomes from Proven Mosaic Embryos Which Were Transferred
0
5
10
15
20
25
30
35
40
<3535+
% o
f e
mb
ryo
s tr
ansf
err
ed
Clincial Ongoing
• Identified by sequential biopsy
• N=98
• Results were unknown at time of transfer
• Follow up with DNA fingerprinting to assure accurate assignment of outcome
How much effort to attain the deliveries of Louise Brown & Elizabeth Carr?
40th attempt was ectopic
104th attempt Louise was born 41 natural cycles
54th attempt Elizabeth was born
13th attempt with hMG
Natural Conceptions: Incidence of Trisomy
Adapted from Hassold T & Hunt P, Nat Rev Genet, 2001
0
10
20
30
40
50
18 23 28 33 38 43
Inci
de
nce
of
Tris
om
y
(% o
f cl
inic
ally
re
cogn
ize
d p
regn
anci
es)
Age
Rate of Aneuploidy in Natural Conceptions and
Embryos from Stimulated Cycles Stratified by Age
0
10
20
30
40
50
60
70
80
90
100
19 21 23 25 27 29 31 33 35 37 39 41 43 45
% A
ne
up
loid
Age
Natural Conceptions: Incidence ofTrisomy in Miscarriage Specimens
Stimulated Cycles: Incidence ofembryonic aneuploidy
0
10
20
30
40
50
60
70
80
90
100
19 21 23 25 27 29 31 33 35 37 39 41 43 45
% A
ne
up
loid
Age
Natural Conceptions: Incidence ofTrisomy in Miscarriage Specimens
Stimulated Cycles: Incidence ofembryonic aneuploidy
0
10
20
30
40
50
60
70
80
90
100
19 21 23 25 27 29 31 33 35 37 39 41 43 45
% A
ne
up
loid
Age
Natural Conceptions: Incidence ofTrisomy in Miscarriage Specimens
Stimulated Cycles: Incidence ofembryonic aneuploidy
Rate of Aneuploidy in Natural Conceptions and
Embryos from Stimulated Cycles Stratified by Age
0
10
20
30
40
50
60
70
80
90
100
19 21 23 25 27 29 31 33 35 37 39 41 43 45
% A
ne
up
loid
Age
Natural Conceptions: Incidence ofTrisomy in Miscarriage Specimens
Stimulated Cycles: Incidence ofembryonic aneuploidy
Superovulation versus Spontaneous Conception: Risk of Aneuploidy in the Products
of Conception Following Loss
0
10
20
30
40
50
60
70
80
90
100
no fertilitymedications
1 cycle after hMG orCC
hMG or CC taken inconception cycle
≥2 cycles after hMG or CC
Pe
rce
nta
ge o
f ch
rom
oso
mal
an
om
alie
s
Boué JG and Boué A, Lancet, 1973
Superovulation vs. Mild Stimulation: Embryonic Aneuploidy Risk
• Prospective RCT
• Mild Stimulation (N=67) – GnRH Antagonist with 150 FSH
– 45% aneuploidy
• Conventional Stimulation (N=44) – GnRH agonist down regulation with 225 FSH
– 63% aneuploidy
• PGD: Blastomere Biopsy and FISH analysis
Baart EB et al, Hum Reprod, 2007
Minimal Stimulation: Miscarriage Risk
• Retrospective Review • Minimal Stimulation
– Clomid, day 3 – HMG/FSH, day 8 qod – Oocyte Maturation:
GnRHa
• 6,549 pregnancies conceived after minimal stimulation
• Rate of miscarriage may
reflect age-related increase in aneuploidy
Teramoto & Kato, RBMO, 2007
16.7%
23.5%
33.9%
46.9%
0%
10%
20%
30%
40%
50%
60%
≤35 years 36-38 years 39-41 years >41 years
Ris
k o
f lo
ss
International Society for Mild Approaches in Assisted Reproduction: Standardized Terminology
• Natural Cycle IVF – Spontaneous cycle, no additional medications – Aim for single oocyte
• Modified Natural Cycle IVF
– Spontaneous cycle with additional medications • hCG, GnRH antagonist +/- FSH or HMG add-back, NSAIDs
– Aim for single oocyte with decreased risk of cancellation
• Mild IVF
– Lower doses and/or shorter duration of FSH or HMG with GnRH antagonist co-treatment or:
– Oral compounds +/- FSH or HMG – Aim for 2-7 oocytes
Nargand G et al, Hum Reprod, 2007
Natural Cycle IVF Study
• Inclusion Criteria – Age 18-49 with regular ovulatory cycles – No medical contraindications to treatment
• Spontaneous cycle – No exogenous medications to induced ovulation utilized – (i.e. no LH, FSH, GnRH antagonist or NSAID)
– Final Oocyte Maturation • induced when follicle ≥15mm (mean of ~18 mm) • Dual Trigger: hCG and GnRH agonist
Conventional IVF: Control Population
• Retrospective Cohort
– 15,169 embryos
– Franasiak 2014
• Inclusion Criteria: Infertile patients undergoing IVF cycles with CCS
• Age Range: 22-49 years old
Results: Natural Cycle IVF
• 173 embryos obtained from natural IVF cycles
– 99 Usable Blastocysts + 74 Arrested Embryos
– 53.2% (N=90) of all embryos were aneuploid
• 99 Usable Blastocysts
– 40.4% (N=40) were aneuploid
Aneuploidy rate by maternal age: n=173 embryos: usable blasts & arrested
0
10
20
30
40
50
60
70
80
90
100
<31 31-34 35-37 38-40 41-42 >42
Pro
po
rtio
n o
f e
mb
ryo
s w
ith
an
eu
plo
idy
(%)
Patient age (years)
Natural
Superovulation
P=NS
Aneuploidy rate by maternal age: n=99 usable blasts
0
10
20
30
40
50
60
70
80
90
100
<31 31-34 35-37 38-40 41-42 >42
Pro
po
rtio
n o
f e
mb
ryo
s w
ith
an
eu
plo
idy
(%)
Patient age (years)
Natural
Superovulation
P=NS
Embryonic Aneuploidy in Stimulated and Natural Cycles
• Embryonic aneuploidy rates in natural and
stimulated IVF cycles do not differ.
• These data do not support a causative role for gonadotropin superovulation in embryonic aneuploidy.
Rate of Aneuploidy in Natural Conceptions and
Embryos from Stimulated Cycles Stratified by Age
0
10
20
30
40
50
60
70
80
90
100
19 21 23 25 27 29 31 33 35 37 39 41 43 45
% A
ne
up
loid
Age
Natural Conceptions: Incidence ofTrisomy in Miscarriage Specimens
Stimulated Cycles: Incidence ofembryonic aneuploidy
Clinical and Scientific Director Richard T. Scott
Physicians Paul Bergh
Michael Bohrer Maria Costantini Michael Drews
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The Treff Laboratory Nathan Treff
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