1. PGS with fresh embryo transfer
Fresh embryo
Santiago Munné, PhD
Reprogenetics Laboratories
US: Livingston (NJ), Los Angeles (CA), Chicago (IL), Portland (OR), Boca Raton (FL) / Europe:
Barcelona (Spain), Oxford (UK), Hamburg (Germany) / Asia: Kobe (Japan), Macao, Abu Dhabi
(UAE) / Latin America: Lima (Peru), Buenos Aires (Argentina), Sao Paulo (Brazil), DF (Mexico)
3. Most loss of implantation is caused
by chromosome abnormalities
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
<35 35-37 38-40 41-42
% euploid no imp
% euploid implan
% aneuploid
Reprogenetics data: 96 centers, >3500 cycles, >19,000 blastocysts analyzed by aCGH to 9/2013
4. Euploidy decreases with age but
not with cohort size
# of
blastocysts
% normal embryos
egg
donors
<35
years
35-37
years
38-40
years
41-42
years
N = 4,747 cycles and 29,803 embryos, up to 12/2013.
Ata, Munne et al. (2012) Reprod Biomed Online and unpublished data.
>42
years
1-3 58% 61% 51% 39% 22% 13%
4-6 62% 60% 52% 38% 23% 17%
7-10 65% 62% 51% 36% 21% 14%
>10 68% 63% 55% 37% 25% n/a
5. The PGS hypothesis - updated
• 50% of blastocysts are aneuploid,
• Aneuploidy increases with maternal age
• Maternal age is inversely proportional to implantation
• The error rate of PGS with CCS is low (<2%)
• And blastocyst biopsy is non-detrimental
Therefore PGS with CCS and blastocyst biopsy:
• Improves Implantation rates
• Eliminates the Maternal age effect on implantation
CCS: comprehensive chromosome analysis, such as array CGH, qPCR, Karyomaping, NGS
7. Biopsy stage:
before arrays
• 30% postmeiotic
abnormalities undetected
• Triple amount of cells to
analyze vs. blastocysts
•30%a to 60%b loss of
implantation potential
• PGD can compensate
the damage but does
not reach its potential
• Centers not proficient in
blastocyst culture until recently
• Applied clinically in 2005 c
• First positive results in 2010 d
a Scott et al (2013), b Mastenbroek et al. (2007), c McArthur et al. (2005), d Schoolcraft et al. (2010)
8. blastocyst biopsy:
Advantages
Advantages:
• More DNA: less no results
• Less mosaicism = low error rate
• Reduced impact of embryo biopsy
• Less embryos to process
• Facilitates single embryo transfer
• Frozen cycle: Uterine environment
optimized after thaw
Disadvantages:
• Not all embryos reach blastocyst the same day
9. Evolution of PGS:
Reprogenetics data
7000
6000
5000
4000
3000
2000
1000
0
2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013
Number of Cases
Year
aCGH day 5
aCGH day 3
CGH
FISH 12
FISH 5-9
Reprogenetics Laboratories: 42,000 PGD procedures up to 3/2014
11. aCGH advantages
• All 24 chromosome aneuploidies and translocations
detected.
• Results in <16 hours:
allows for day 5 biopsy and 10am day 6 transfer
• Parental DNA not required: ad hoc decisions possible.
• ICSI not required.
13. aCGH validation:
reanalysis of blastocysts
Reanalysis
method
Confirmed
Euploid
Confirmed
abnormal
Fragouli et al 2011 FISH, aCGH 23/25 27/27 50/52
Capalbo et al. 2013 FISH 19/20 50/50 69/70
Colls et al. 2013 FISH, aCGH 7/7 39/40 46/47
Wells et al. 2013 Next Gen.
Sequencing
23/23 67/67 90/90
Total 96%
Sensitivity
99.5%
Specificity
11% of embryos were mosaic, explaining the 2.4% error rate
TOTAL
1.6%
Error rate
Fragouli et al. (2011) Hum. Reprod. 26: 480-90, Colls et al. (2013) ASRM P-168,
Capalbo et al. (2013) Hum Reprod, in press, Wells et al. (2013) ASRM O-435 and
unpublished data from Reprogenetics
14. Speed
of different techniques
Biopsy Reception Results by
qPCR: day 5 day 5, 6pm day 6, am
aCGH: day 5 day 5, 6pm day 6, noon
NGS: day 5 day 5, 6pm day 6, noon
SNPs: day 5 day 5, noon day 6, 6pm
15. Differences between PGD methods
PCR PCR + SNP Next Gen
aCGH arrays* Sequencing
Detects aneuploidy no yes yes yes
Detects gene defects yes yes yes yes
Detects mitotic errors no yes no yes
>2 month of Preparation yes yes no no
Requires affected proband no no yes no
# genomes / run 0 0 0 1 *
* Karyomapping using BlueGnome
** with NextSeq
16. NGS:
Capacity vs. Depth
samples genome depth of
/ run coverage coverage
Genome 1 100% x 30
PGS * 16- 96 ≤ 10% x1 to x3
Wells, Kaur, Rico, Grifo, Anderson, Sherlock, Taylor, Munne (2013) ESHRE, Yin et al (2013) Biol Reprod 88, 69
* Output: MiSeq = PGM << NextSeq
17. validation of
Next Generation Sequencing (NGS)
78 blastocysts previously diagnosed by aCGH were reanalyzed
by NGS in a blinded experiment.
21/21 euploid
90/90 aneuploid
> 99% concordant
Allen Kung et al. (2014) ESHRE and Reprogenetics unpublished data
19. Meta analysis of RCTs
IMPLANTATION RATES IN RCT STUDIES USING PGS v2:
Control PGS
Yang et al. 2012 46% 69%
Scott et al. 2013 63% 80%
Forman et al. 2013 40% 58%
TOTAL 53% 73% P<0.001
21. aCGH eliminates the negative effect of
maternal age on implantation
60%
50%
40%
30%
20%
10%
0%
<35 35-37 39-40 41-42 >42
No PGS *
SART
bPlGasSto (cayCsGt H) **
Implantation rate
Maternal age
n/a
* SART 2011
** Harton, Munné et al. (2013) Fertil Steril. And unpublished data to 8/2013. N >800 blast biopsies
22. Miscarriage rate after
blastocyst biopsy
Compared to SART: Compared to other studies:
40%
35%
30%
25%
20%
15%
10%
5%
0%
No PGS *
PGS **
<35 35-37 38-40 41-42
Preg
nan
cies
age SAB
This
study
307 34.9 7.5%
Scott et
al. 2013
72 32.2 8.3%
*SART, ** Harton et al. (2013) Fertil Steril, and unpublished data
24. In embryo banking aneuploidy rates
Remain constant
1st
cycle
Reprogenetics data, unpublished
>350 cycles of embryo banking, average age 39.9
2nd
cycle
3rd
cycle
Total
Euploidy rate 29% 29% 27% 28%
# blastocyst produced 2.4 3.0 2.5 6.3
# euploid blastocysts 0.7 0.9 0.7 2.2
25. Embryo banking for low responders
# of
blastocysts
or bad prognosis patients
% of patients with normal embryos
egg
donors
<35
years
35-37
years
38-40
years
41-42
years
N = 3,571 cycles and 19,356 embryos, up to 8/2013.
Ata, Munne et al. (2012) Reprod Biomed Online and unpublished data.
>42
years
1-3 86% 85% 72% 60% 58% 24%
4-6 95% 97% 95% 88% 69% 54%
7-10 100% 99% 96% 92% 85% 65%
>7-10 100% 100% 98% 98% 92% 83%
26. Success of SET by Euploid Cohort Size
26
No.
Euploid
Embryos
CPR
1 23/55 (41.8%)
2 13/27 (48.1%)
3 9/19 (47.4%)
4 16/21 (76.2%)
5 8/11 (72.7%)
6 6/8 (75.0%)
>7 11/15 (78.6%)
p < 0.01
S. Morin, K. Melzer, J. Grifo, P. Colls, Z. Zheng, S. Munné (2014) JARG
27. Cycles needed to accumulate
3 euploid blasts
Age Av. blastocysts Av. euploid
# cycles to
achieve 95%
patients w/ 3
euploid
35-39 5.2 2.5 2.7
40-42 4.2 1.1 5.8
28. Advantages of embryo banking
• Less patient “fatigue”: less drop out from
cycle to cycle.
• Cheaper PGD: One fee per package of IVF
cycles
• Facilitates “guaranteed baby” plans
30. Does COS affects endometrial receptivity?
Roque et al., 2012
RR=1.31 [1.10-1.56]
Meta-Analysis
Courtesy: Dr. Racowsky
Non-PGD Studies
31. Does COS affects endometrial receptivity?
The only
effect is on
embryos
developing to
blastocyst on
day 6, which
in PGS with
fresh transfer
they are
frozen.
Non-PGD Studies
32. Is it worthy to biopsy
day 6 blastocysts?
The differences between day 5 biopsy and fresh transfer vs. day 5-6
biopsy and vitrification is that the later includes day 6 biopsies:
- Day-5 morulas were cultured to day-6 and biopsied if reached blastocyst
- SET of blastocysts either biopsied on day 5 or on day 6, thawed transfer
Day 5
blastocyst
Day 6
blastocyst
Implantation 61% 60% N.S.
Euploidy 56% 42% P<0.025
Reprogenetics, unpublished
33. Controlled studies
comparing both treatments
SET SET SET
fresh FET FET
Control Control PGS-CCS
Cycles 118 272 347
Maternal age 36.1 36.8 37.9 p<0.05
Implantation rate (FHT) 49.2% 52.6% 65.1% p<0.05
SAB 14.3% 14.4% 4.6% p<0.05
Ongoing pregnancy rate 40.7% 43.8% 60.0% p<0.05
Difference between control and PGS:
not due to FET
Schoolcraft et al. Fertil Steril 2013;100:615–9
34. Meta-analysis
# cycles
Ongoing
pregnancy rate /
transfer
FET
fresh
day 6 FET
fresh
day 6
Anderson et al. 2013 38 46 55% 66% NS
Hill et al. 2013 143 99 56% 45% NS
Forman et al. 2013 27 62 55% 65% NS
Total 208 207 56% 56% NS
Anderson et al. (2013) ASRM O-120, Barrit et al. (2013) ASRM abstract P-167,
Forman et al. (2013) Fertil Steril
35. day 6 transfer and the
window of implantation
No studies published, but some recommend:
• Replace before noon day 6 but…
• If last progesterone before trigger is elevated then
freeze: the endometrium is advanced and the window of
implantation could be missed.
• Each center needs to determine their threshold. One
center recommends 1.5 or over to freeze.
36. What IVF centers are
doing in the US?
2013 2014 Q1
Day 3 fresh 18.1% 20.9%
Day 5 fresh 14.3% 12.4%
Day 5 frozen 67.5% 66.6%
Total PGD
4359 1460
procedures
In Germany is mostly PB biopsy, and in Spain mostly day 3
37. Fresh: pros and cons
Pros
- Day 5 blastocysts implant equally well on d6 or FET
- Lower cost (no freezing)
- Some centers biopsy day 5 morulas
- International patients: no need to come back for transfer
- INSTANT SATISFACTION
Cons
- Day 6 blastocysts need to be frozen anyway
- Does not allow for embryo banking
38. Frozen: pros and cons
Pros
- Day 5 blastocysts implant equally well on d6 or FET
- Day 6 blastocysts implant better frozen
- Allows for embryo banking for poor prognosis patients
- Better uterine receptivity in aggressive stimulations
Cons
- Higher cost (freezing)
- International patients need to come back for transfer
- Delayed gratification
39. Ongoing RCT:
Fresh vs. frozen using NGS
Procedure:
all patients undergo blastocyst biopsy and analysis by Next
Gen Sequencing.
Group comparison:
Patients randomized to day 6 transfer or to FET.
Location:
Oregon Reproductive Medicine, Portland, OR.
40. munne@reprogenetics.com
www.reprogenetics.com
Reprogenetics
Scientists
Jacques Cohen, PhD (US)
Santiago Munne, PhD (US)
Dagan Wells, PhD (UK)
Renata Prates (US)
Samer Alfarawati (UK)
Souraya Jaroudi (UAE)
Tomas Escudero (US)
Mireia Sandalinas, PhD (Spain)
Luis Guzman, PhD (Peru)
J. Horcajadas, PhD (Latin Am.)
M. Konstantinidis, PhD (US)
N’Neka Goodall (US)
Allen Kung (US)
Lia Ribustello (US)
Lab & Medical Directors
Pere Colls, PhD (US)
Carles Gimenez, PhD (Spain)
Elpida Fragouli, PhD (UK)
Karsten Held, MD (Germany)
Tetsuo Otani, MD (Japan)
Muriel Roche, PhD (Japan)
Braulio Peramo, MD (UAE)
Ahmed Yesilyurt, MD (Turkey)
Xuezhong Zeng, MD (China)
Francisco Rocha (Mexico)
Embryologists
Kelly Ketterson
Catherine Welch
Tim Schimmel
Genetic Councilors
Jill Fischer
Amy Jordan
Erin Mills
G. Manassero, MD
