This document summarizes research on preimplantation genetic diagnosis (PGD) and the use of array comparative genomic hybridization (aCGH) to detect chromosome abnormalities in embryos. Key points include: 1) Over 50% of embryos, especially in older women, have chromosome abnormalities detectable by aCGH. 2) Blastocyst biopsy and aCGH analysis results in high implantation rates of over 75% that are close to the predicted maximum and become independent of maternal age. 3) Studies showing improved outcomes with PGD have low error rates, experience, and appropriately select high-risk patient populations.

1. PGD and implantation
USA: Europe: Asia: South America:
Livingston, NJ Barcelona, Spain Kobe, Japan Lima, Peru
Oxford, UK
Hamburg, Germany
Santiago Munné, Ph.D.

2. High rates of
Chromosome abnormalities

3. embryos analyzed: 6054. Morphologically normal embryos: 3751. Source: Munné et al. 2007.
Similar results also found by Munne et al 1995, Marquez et al. 2000, Magli et al. 2007.
% chromosomally
abnormal embryos
56%
Maternal age
Morphology:
The majority of embryos with ‘good’
morphology are chromosomally abnormal

4. <35 35-37 38-40 41-42
- Implantation * 34% 25% 17% 9%
- spontaneous losses * 9% 18% 27% 40%
- Ongoing implantation ** 30% 20% 12% 6%
- Normal embryos (array CGH) *** 46% 33% 25% 18%
- Loss of implantation not explained
by chromosome abnormalities 16% 13% 13% 12%
* SART (2008), ** estimated from SART (2008), *** Reprogenetics
IVF results (SART 2008) compared
to chromosome abnormalities

5. transfer
transfer
PGD + morphology selection chose differently:
Best morphology
Abnormal by PGD

6. PGD
Hypothesis
PGD may improve ART outcome in women of
advanced maternal age Munné et al. (1993)
Despite large studies indicating the advantages
of aneuploidy screening, the notion that PGS for
infertility is beneficial is not shared uniformly.

7. Contradicting PGD results
using day 3 biopsy and FISH
Positive effect
Gianaroli et al. 1999
Munne et al 1999
Gianaroli et al 2001a
Gianaroli et al. 2001b
Munne et al. 2003
Gianaroli et al. 2004
Munne et al. 2005
Munne et al 2006
Verlinsky et al. 2005
Colls et al. 2007
Garrisi et al. 2009
Rubio et al. 2009
No effect (small)
Werlin et al. 2003
Jansen et al. 2008
Mersereau et al. 2008
Schoolcraft et al. 2009
No effect (Large)
Staessen et al. 2004
Platteau et al. 2005
Negative effect
Mastenbroek et al. 2007
Hardarson et al. 2008

8. Explanation for contradicting PGD results
1) Biological: Mosaicism
2) Genetic technique: FISH error rate
3) Biopsy technique: Embryo damage

9. Mosaicism

10. 592 embryos found abnormal by PGD were reanalyzed in all cells and found
to be:
normal 13
mosaic <49% abnormal 27
mosaic 50-99% abnormal 124
mosaic 100% abnormal 297
homogeneously abnormal 131
Colls et al. (2007)
Mosaicism produces 7% misdiagnosis
1[13]1[16]2[18]2[21]1[22]
2[13]1[16]2[18]2[21]2[22]
1[13]1[16]2[18]2[21]1[22]
1[16] 2[13]2[16]2[18]2[21]2[22]
2[13]1[16]2[18]1[21]1[22]
2[13]3[18]1[21]1[22]
3[13]1[16]2[18]1[21]3[22]
1[13]1[16]1[18]1[21]1[22]
3[13]1[16]2[18]1[21]3[22]
7%

11. Day 3 embryo biopsy

12. Advantages / disadvantages
day 3 biopsy
Advantages:
30% more abnormalities detected than PB biopsy
Cells undifferentiated
Disadvantages:
1/6 cells = 17% of body mass
1/8 cells = 12.5% body mass
Cell polarization?
More sensitive to biopsy damage?

13. “ The data presented here clearly indicates that two cell
biopsy significantly impacts clinical outcome. Our
previous report providing no arguments in favor of PGS
(Staessen et al., 2004) was criticized by others arguing
that PGS might have been beneficial if only one cell had
been removed (Cohen et al., 2007). In respect to the
present findings, this criticism seems justified”.
P < 0.001
Two cell biopsy is detrimental

14. 1) 20% of cycles undiagnosed (literature: 1-3% of embryos *)
2) 59% implantation reduction due to biopsy:
59% reduction
implantation
Control 14.7%
Biopsied, no PGD 6.0%
Biopsied and PGD 16.8%
* 1% Gianaroli et al. (2004), 3.1% Colls et al. (2007)
Effect of biopsy in
Mastenbroek et al. (2007)

15. FISH technique

16. Analysis of remaining cells of embryos previously analyzed by PGD:
study technique error rate
Baart et al 2004 FISH 50.0%
Li et al. 2005 FISH 40.0%
Gleicher et al. 2009 FISH 15-20%
Munne et al. 2002 FISH-9 7.2%
Colls et al., 2007 FISH-9 4.7%
Magli et al. 2007 FISH-9 3.7%
Gutierrez et al. 2010 array CGH 1.8%
Error rate should be <10%

17. Aneuploidy rates for chromosomes X,Y,13,18,21. Munne et al. 2006 and Reprogenetics data up to
10/2007. Average age 37, Observed: Based on 2300 pregnancies after PGD, Expected: Eiben et al.
1994. Observed and expected adjusted by maternal ages
2.60%
0.50%
0.00%
0.50%
1.00%
1.50%
2.00%
2.50%
3.00%
trisomic conceptions
expected
observed
P<0.001
Significant reduction in
Trisomic offspring

18. New approach to PGD:
• 24 chromosome analysis
by arrays
• Blastocyst biopsy and
vitrification

19. Array CGH
Test
DNA
Normal
DNA
4000 probes
Same band resolution as karyotype

20. 46,XY

21. 47,XY+2

22. aCGH advantages
• All 24 chromosome type of aneuploidies detected
• Results in 24 hours; allows for PB or day 3 biopsy
• Parental DNA not required: ad hoc decisions possible
• Used in >15,000 patients with mental retardation

23. 46,XX-10 +16
aCGH detected 50% more abnormalities than FISH-12 and 20%
more abnormal embryos (Colls et al. 2009)
Detection of abnormalities:
aCGH vs. FISH-12
Detectable by FISH

24. aCGH validation: no results
Embryos undiagnosed:
biopsy on day 3: 2% (16/724)
biopsy on day 5: ≈ 0% (0/64)
Gutierrez-Mateo et al. (in press)

25. aCGH validation: error rate
• Validation method 1: single cells from cell lines analyzed*
Error rate in euploid and aneuploid cell lines: 0%
• Validation method 2: Reanalysis of the rest of the embryo
by FISH with 19 chromosomes probes**
Error rate from day 3 biopsies: 3%
*Mamas et al (submitted), **Gutierrez et al. (in press) and further
analysis at Reprogenetics

26. aCGH vs. SNP arrays: Genome coverage
# of probe genome
probes size covered
aCGH 4,000 x 150,000 kb = 600.0 Mb (25%)
SNPs 300,000 x 50 kb = 1.5 Mb (>0.1%)

27. Comparison of platforms
FISH-12 array SNP
probes a CGH a arrays
Day 3 biopsy/ day 5 results yes yes yes
Parental DNA needed no no yes
Aneuploid embryos detected 89% 100% 97%
Detect gene defects no yes-split yes
Detect polyploidy (errors) yes (1.8% ) yes
Detect MII trisomies w/o crossover yes yes no if qual.
Detect mitotic trisomies (errors) yes yes (3%)
Error rate (cell lines analysis) 2% 0% d 4% b
Error rate (embryo reanalysis) 7% 3% e unk
Embryos with no results 3% 2% e 8% b
Increased implantation rate (day 3) + + unk
Increased implantation rate (day 5) unk + + c
aReprogenetics, bJonhson et al. (2010), cScott et al. (2010), dMamas et al (in press), eGutierrez-Mateo (in press)

28. Day 3 biopsy, day 5 transfer
and array CGH
Cycles performed: 303
Maternal age (av.) 37.2
Pregnancy Rate Ongoing Pregnancy Rate
Per Cycle Per ET Per Cycle Per ET
Control 38% 38% 31% 31%
PGD 46% 59% 42% 54%
NS < 0.001 NS < 0.001
* Expected from each center SART data, controlled by age
Data from 24 centers. Munné et al. (2010) PGDIS, and unpublished data

29. Results with array CGH for RPL
Indication: ≥2 previous miscarriages
Procedure: Biopsy on day 3, PGD by array CGH
Population: average 36 years old, Av. 3.2 previous miscarriages
Observed:
29/53 transfers resulted in pregnancy (55%)
41/82 embryos replaced implanted (50%)
2/29 pregnancies miscarried (7%)
Expected:
Miscarriage rate (Brigham et al. 1999) for this population: 30%

30. Prognosis depending on
age and ovarian response
<35
years old
35-39
years old
40-42
years old
>42
years old
20% 53% 47% 89%
50% 16% 28% 4%
4% 18% 33% 75%
43% 30% 17% 7%
0% 11% 26% 29%
47% 30% 22% 11%
0% 0% 6% 20%
48% 33% 21% 9%
> 10
(% normal embryos)
% patients with all embryos abnormal
5-7
8-10
# day 3
embryos
1-4

31. Patients with good prognosis with PGD
# day 3
embryos
% patients with all embryos abnormal
(% normal embryos)
<35 years
old
35-39
years old
40-42
years old
>42
years old
1-4 20% 53% 47% 89%
50% 16% 28% 4%
5-7 4% 18% 33% 75%
43% 30% 17% 7%
8-10 0% 11% 26% 29%
47% 30% 22% 11%
> 10 0% 0% 6% 20%
48% 33% 21% 9%

32. “closure” indication:
PGD to transition to egg donation
# day 3
embryos
% patients with all embryos abnormal
(% normal embryos)
<35 years
old
35-39
years old
40-42
years old
>42
years old
1-4 20% 53% 47% 89%
50% 16% 28% 4%
5-7 4% 18% 33% 75%
43% 30% 17% 7%
8-10 0% 11% 26% 29%
47% 30% 22% 11%
> 10 0% 0% 6% 20%
48% 33% 21% 9%

33. Results of aCGH in PB and day 3 biopsies
ESHRE study: PB data
Average age 40
Cycles 42
Embryo replaced n/a
Implantation rate n/a
Pregnancy rate 19%
Error rate 11%
Reprogenetics: data day 3 biopsy
Average age 40
Cycles 107
Av. Embryos replaced 1.0
Implantation rate 31%
Pregnancy rate 26%
Error rate 2% *
* Gutierrez-Mateo et al., Fertil Steril,
accepted

34. Array CGH and
Blastocyst biopsy

35. array CGH on blastocyst biopsies:
Why?
Advantages:
1) More DNA: More robust diagnosis
2) Eliminates some mosaic embryos
3) Reduces error rate
4) Reduced impact of embryo biopsy
5) Less embryos to process
6) Facilitates single embryo transfer
7) Uterine environment optimized after thaw

36. Effect of embryo vitrification and
Replacement on next cycle

37. Cycles Mat. Prev. embryos implant. ongoing
age failed replaced (+ sac) preg.
cycles
CGH : 45 37.7 2.4 2.0 67% 79%
control : 113 37.1 1.2 2.7 28% 60%
p=0.0003
Schoolcraft et al. (in press)
CGH on blastocyst biopsies:
clinical results

38. aCGH with biopsy on day 3 or day 5:
Results from 22 centers
day 3 day 5
biopsy biopsy
Maternal age: 37.6 38.2
# zygotes 8.6 8.9
% normal embryos: 35% 36%
cycles: 152 26
Implantation 42% 77%
Pregnancies / pick up 42% 50%
Pregnancies / transfer 54% 73%
Miscarriage rate 6% 0%
Biopsy
Effect?

39. <35 35-37 38-40 41-42
- Ongoing implantation w/o PGD ** 30% 20% 12% 6%
- Normal embryos (array CGH) *** 46% 33% 25% 18%
- Loss of implantation not explained
by chromosome abnormalities 16% 13% 13% 12%
- Maximum ongoing implantation
with PGD 84% 87% 87% 88%
** estimated from SART (2008), *** Reprogenetics
77% implantation is close to the
Maximum predicted

40. 0
10
20
30
40
50
60
70
80
90
30-34 35-38 39-42 43-45
implantation rate
per replaced
embryo
aneuploidy rate
cycles with all
embryos abnormal
After CGH on blastocysts Implantation
becomes independent of age
For day 3 biopsy, the % of cycles with all abnormal embryos is higher

41. CONCLUSIONS

42. A majority of embryos at any age have numerical
chromosome abnormalities (54% in <35 to 82% in
>40 y. o.).
Other factors may contribute to only 20% of lost
ongoing implantation potential
Conclusions: Chromosome abnormalities

43. - Mosaics are common but the majority of
them have >50% abnormal cells
- This results in less than 7% of errors
- Technical errors in laboratories starting to
offer PGD should not be confused with
mosaics
- There is extremely low self correction of
abnormal to normal embryos in vivo
Conclusions: Mosaicism not an issue

44. Studies with improved results differ from those that show
no improvement in that:
1) Reduce biopsy damage (1 cell, experience, blast?)
2) Low error rate (fixation, NRR, 2 analyzers)
3) Analyze 16,15,21,22 chromosomes + ≥4 more
4) Extensive experience
5) Appropriate population (≥5 embryos, ≥ 35 y. old)
Conclusions: FISH studies

45. With day 3 biopsy:
- Only 2% error rate
- Preliminary results suggest significant implantation
improvement and reduction in miscarriages
With day 5 biopsy and vitrification:
- 0% error rate
- 67-80% implantation rate
- Optimal endometrium recpetivity
Conclusions: array CGH

46. Santiago Munné, PhD, Director
Jacques Cohen, PhD, Director
munne@reprogenetics.com
www.reprogenetics.com
Pere Colls, Ph.D
Dagan Wells, Ph.D
George Pieczenik, Ph.D
Cristina Gutiérrez, Ph.D
Jorge Sanchez, PhD
John Zheng, MD
Tomas Escudero, MS
Kelly Ketterson, MS
Jill Fischer, MS
Gary Harton, MS
Jessica Vega, MS
Tim Schimmel
Sasha Sadowy
Sophia Tormasi
N-neka Goodall
Renata Prates
Piedad Garzón
Laurie Ferrara
Bekka Sellon-Wright
Maria Feldhaus
USA
Spain
Mireia Sandalinas, MS
Carles Giménez, PhD
César Arjona, MS
Ana Jiménez, PhD
Elena Garcia, MS
Japan
Tetsuo Otani, MD
Muriel Roche
Miho Mizuike
UK
Dagan Wells, PhD
Elpida Fragouli, PhD
Samer Alfarawati, MS
Michalis Konstantinidis
South America
Paul Lopez
Luis Alberto Guzman
Germany
Karsten Held, MD