This document discusses various methods for preimplantation genetic diagnosis (PGD) using embryo biopsy and genetic analysis. It finds that a high percentage (over 50%) of morphologically normal embryos are chromosomally abnormal, and this percentage increases with maternal age. New methods like array comparative genomic hybridization (aCGH) allow analysis of all 24 chromosomes with a low error rate (<2%) and without need for parental samples. Clinical trials show improved pregnancy and implantation rates using aCGH with day 3 or day 5 embryo biopsy compared to no PGD. Blastocyst biopsy provides more DNA for analysis and may reduce mosaicism effects. Overall, the document evaluates different PGD methods and finds aCGH with blastoc

1. Genomics for embryos selection
USA: Europe: Asia: South America:
Livingston, NJ Barcelona, Spain Kobe, Japan Lima, Peru
Los Angeles, CA 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
- Ongoing implantation without PGD * 30% 20% 12% 6%
- Abnormal embryos (array CGH) ** 54% 67% 75% 82%
- Non-chromosomal implantation loss 16% 13% 13% 12%
*estimated from SART (2008), ** Reprogenetics
IVF Results (SART 2008) Compared
to Chromosome Abnormalities

5. Conflicting 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
Werlin et al. 2003
Staessen et al. 2004
Platteau et al. 2005
Jansen et al. 2008
Mersereau et al. 2008
Schoolcraft et al. 2009
Negative effect
Mastenbroek et al. 2007
Hardarson et al. 2008

6. Optimal PGD Questionable PGD
Biopsy media with aminoacids simple media
Biopsy time / embryo 1 min > 5 min
# cells biopsied one two
Fixation method Carnoy’s Tween 20
# chromosomes tested ≥8 6
# analysts / case 2 1
Reanalysis no results yes no
Large experience yes no
Error rate <10% 10-50%
Number of zygotes >5  5
Reviewed in Munné et al. (2010) Fertil. Steril. 94:408
Optimal PGD Methods for FISH

7. 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%
Munne et al. 2010 array CGH 1.8%
Technical Errors Should be <10%

8. • 24 chromosomes analysis: array CGH, SNP arrays, qPCR
• Blastocyst biopsy
• vitrification
New developments

9. 24-chromosme analysis:
• array CGH
• SNP arrays
• q PCR

10. aCGH vs. SNP arrays: Resolution
• Abnormalities smaller
than 5MB are not well
understood and difficult to
counsel
• At 150 Kb resolution 50%
of people have CNVs: it is
difficult to differentiate a
new polymorphism from
an abnormality
More resolution, more counseling nightmares:
Array CGH*
Array CGH**
* Bac array, ** oligo array

11. Array CGH
Test
DNA
Normal
DNA
4000 probes
More resolution than karyotype
46,XY

12. 46,XY

13. 46,XX-10 +16

14. Array CGH 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 for
day 3 or 5 biopsies.
• ICSI not required.
• Used extensively in postnatal testing and soon in prenatal
(NIH large study).

15. PGD Reanalysis
Diagnosis: Trisomy 18 Mosaic 100% abnormal
Confirmed?:
Exact abnormality: No
Abnormal embryo: Yes
Definition of error

16. Array CGH Validation Using Cell Lines
Analysis of single cells from euploid and aneuploid cell lines
Array CGH* SNP arrays**
False negatives: 0% 2.1%
False positives: 0% 3.9%
(This method does not account for mosaicism errors.)
*Mamas et al (submitted), **Johnson et al. (2010)

17. aCGH validation:
reanalysis by FISH
- Reanalysis of the remainder cells of the embryo by FISH.
- This detects mosaicism and intrinsic technical errors.
Tris 16
Mono 22
Day-3 Day-5
PGD results not confirmed: 3% (3/104) 0% (0/18)
Gutierrez et al. Fertil Steril (2011) and Reprogenetics unpublished data

18. aCGH validation:
Double cell aCGH analysis
- When IVF centers doubt about the presence of a nucleus
they might biopsy two cells.
- This provide us with two cells analyzed / embryo:
Day-3
Abnormal not confirmed: 2.5% (1/49)
Normal not confirmed: 0.0% (0/32)
Total error rate: 1.2%
Reprogenetics data, unpublished

19. aCGH validation:
biopsy stage
Biopsy Embryos Not confirmed
stage reanalyzed (error rate)
ESHRE PGD consortium* PBs 156 11%
Reprogenetics ** day 3 222 2%
Reprogenetics ** day 5 18 0%
*Geraedts et al. (2011) Human Reprod, in press; ** Gutierrez-Mateo et al (2011) Fertil
Steril., and unpublished data

20. 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%)

21. aCGH vs. SNPs: Parental “Support”
SNP arrays at the single cell level produce noisy and incomplete
results and need parental DNA (“parental support”) in order to
interpret the data.
aCGH does not require to test the parents before PGD
Disadvantages:
1) It prevents deciding on day 3 if to do PGD or not based
on the number of embryos.
2) May result in unnecessary cancellation fees.
3) Misleading: Parental Support simply supports the
software analysis of poor SNP calls, not the patients.

22. aCGH vs. SNPs: Origin of Aneuploidy
Meiotic abnormalities:
Although aCGH cannot detect the meiotic origin of the aneuploidies, it is
well known that >90% of them are maternal in origin.
According to a GSN:
- only < 4% of meiotic aneuploidies were paternal in origin.
- 13% of all abnormalities were paternal. Thus 13-4 = 9% were mitotic.
Our opinion:
- Mitotic abnormalities = mosaicism. Mosaicism involucrates
chromosomes at random and its parental origin is irrelevant.
- If a patient has a high rate of paternal mitotic aneuploidy (mosaicism)
diagnosing a “male factor” is incorrect. Recommending sperm donation
is not warranted.

23. aCGH vs. SNPs: Validation Pitfalls
Reporting Precision per Chromosome
chromosome copy number probability
1 2 0.99
2 2 0.99
3 2 0.99
4 2 0.99
5 2 0.99
6 2 0.96
7 2 0.99
8 3 0.98
9 2 0.99
10 2 0.99
11 2 0.99
12 2 0.99
13 2 0.99
14 2 0.99
15 2 0.99
16 2 0.99
17 2 0.95
18 2 0.99
19 2 0.90
20 2 0.99
21 2 0.94
22 2 0.80
sex Chr XY 0.95
P overall = P1 * P2 * P3 …
To calculate the probability of each and every
copy number being correct, you multiply the
probabilities of each result being correct
together
Probability of Assigning the correct Genetic
diagnosis
(Assume that those which are 99% are halfway
between 99 and 100% = 99.5%)
Probability = 54%
Based on actual clinical case done via Qualitative SNP microarray
Adapted from R.T. Scott

24. Polyploidy cannot be detected by aCGH but the majority of them
have also other abnormalities:
• Abnormal embryos fully reanalyzed: 1,416
• polyploid or haploid by PGD and
polyploid or haploid after reanalysis: 140
• Of the above, that had no other aneuploidies: 27 (1.8%)
Missed total abnormalities: 1.8%
aCGH vs SNPs: polyploidy

25. aCGH vs SNPs: mitotic abnormalities
Qualitative SNP arrays cannot detect mitotic trisomies and
meiotic duplications without recombination:
• Abnormal embryos fully reanalyzed: 1,416
• Of the above, that were 100% mosaic with
only trisomies (mitotic trisomies): 53 (3%)
Not diagnosed by qualitative SNP array: 3%

26. aCGH vs. SNPs: Fingerprinting
Fingerprinting can be used to determine which embryo implanted
when more embryos are replaced than those implanting. It
requires testing the fetus or baby and comparing it with the
embryos.
SNP arrays obtains the information from the embryos during the
PGD test.
aCGH produces enough DNA so left over DNA is always stored and
available for fingerprinting when necessary.

27. Clinical Results
Array CGH with Day 3 Biopsy

28. Day 3: Prognosis depending on
age and ovarian response
egg
donors
<35
years old
35-39
years old
40-42
years old
>42
years old
100% 80% 46% 41% 20%
50% 43% 18% 17% 6%
100% 89% 83% 60% 20%
40% 38% 26% 15% 5%
100% 97% 91% 72% 60%
65% 35% 28% 15% 8%
100% 100% 96% 93% 77%
47% 47% 32% 16% 8%
(% normal embryos)
> 10
5-7
8-10
# day 3
embryos
1-4
Reprogenetics data to 2/2011: 609 cycles, 5101 embryos

29. Day 3 Biopsy, Array CGH
with Day 5 Transfer
Cycles performed: 509
Maternal age (av.) 37.1
Pregnancy Rate Ongoing Pregnancy Rate
Per Cycle Per ET Per Cycle Per ET
Control 38% 38% 31% 31%
PGD 38% 54% 35% 52%
NS < 0.001 NS < 0.001
Data from all referring centers. Munné et al. (2010) ASRM, and unpublished data

30. Array CGH for Recurrent Pregnancy Loss
Indication: ≥3 previous miscarriages, <42 years old
Procedure: Biopsy on day 3, PGD by array CGH
Population: average 36 years old, Av. 3.2 previous miscarriages
Expected:
Miscarriage rate (Brigham et al. 1999) for this population: 30%
Observed: <35 years 35-41 years
Cycles 20 49
Pregnancy rate / retrieval 45% 41%
Pregnancy rate / transfer 47% 52%
Miscarriage rate 0% 5%

31. Arrays and
Blastocyst Biopsy

32. Array CGH on Blastocyst Biopsies: Why?
Advantages:
1) More DNA: More robust diagnosis
2) Blastocysts have less mosaicism
3) Lowest error rate (≈0%)
4) Reduced impact of embryo biopsy
5) Less embryos to process
6) Facilitates single embryo transfer
7) If frozen cycle: Uterine environment
may be optimized after thaw

33. Cycles Mat. Prev. embryos implant.
age failed replaced (+ sac)
cycles
CGH : 45 37.7 2.4 2.0 72%
control : 113 37.1 1.2 2.7 46%
p=0.0003
Schoolcraft et al. (2010) Fertil. Steril. 94:1700
CGH, Blastocyst Biopsy and Vitrification
Clinical Results

34. Day 5: Prognosis depending on
age and ovarian response
egg
donors
<35
years old
35-39
years old
40-42
years old
>42
years old
100% 63% 68% 40%
50% n/a 50% 36% 20%
100% 100% 87% 56% 65%
70% 72% 44% 38% 23%
100% 100% 100% 100%
73% 57% 50% 36% n/a
100%
58% n/a n/a n/a n/a
(% normal embryos)
> 10
4-6
7-10
# day 5
embryos
1-3
Reprogenetics data to 2/2011: 109 cycles, 595 embryos

35. Array CGH with Biopsy on Day 3 or Day 5
Results from All Referring Centers
day 3 day 5
biopsy biopsy
Maternal age: 37.1 37.0
# zygotes 8.6 4.6
% normal embryos: 35% 48%
cycles: 509 70
Implantation after PGD 41% 60%
Pregnancies / pick up 40% 46%
Pregnancies / transfer 54% 65%
Biopsy or
Thaw
transfer
Effect

36. Scott et al., 2010 ASRM, O-5 (RCT data)
Treff et al., 2009 ASRM, O-169 (qPCR methods)
Treff et al., 2010 Fertil Steril. 94(6):2017-21 (SNP array methods)
Day 5 biopsy and
SNP array and qPCR PGD
Courtesy of Dr. Treff

37. Day 5 Blastocysts Biopsy and CGH
Abrogate the Maternal Age Effect
Day 5 data: CGH and aCGH data, Day 3 data: from aCGH, *cycles with >4 embryos biopsied
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
Day 5 biopsy
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
<35 35-39 40-42 43-45
implantation rate
aneuploidy rate
cycles with all abnormal
Day 3 biopsy*

38. Arrays and
vitrification

39. Effect of embryo vitrification:
Non-PGD results

40. Array CGH with Biopsy on Day 3 or Day 5
Results from All Referring Centers
day 3 day 5
biopsy biopsy
Maternal age: 37.1 37.0
# zygotes 8.6 4.6
% normal embryos: 35% 48%
cycles: 509 70
Implantation after PGD 41% 60%
Pregnancies / pick up 40% 46%
Pregnancies / transfer 54% 65%
Biopsy or
Thaw
transfer
Effect

41. PGD Av. day day pregnancies
Test age biopsy replacement / transfer
FISH-9 39.6 3 day 5 29%
FISH-9 39.0 3 thaw cycle 47%
FISH-12 39.3 3 thaw cycle 46%
FISH-12 39.5 5 thaw cycle 40%
p<0.05
Effect of embryo vitrification:
FISH data
Reprogenetics-Japan, unpublished data

42. Effect of embryo vitrification:
CGH + SNP array data
- PGD by CGH or SNP array
- PGD and control both frozen cycles:
Control PGD *
Implantation 35% 62.6% p<0.0001
Pregnant 57% 81.4% p<0.001
Schlenker et al. (2010) Fertil Steril

43. Conclusions

44. 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

45. - Mosaics are common but the majority of
them have 100% abnormal cells once
analyzed for all chromosomes.
- This results in less than 3% of errors.
- Error rates are laboratory-dependent and are
the best parameter to evaluate a PGD lab.
Conclusions: Mosaicism not an issue

46. With day 3 biopsy:
- Only 3% error rate with aCGH
- If normal embryos are available ongoing implantation
rates improve.
With day 5 biopsy and vitrification:
- 0% error rate
- 60-80% implantation rate
- Optimal endometrium receptivity
Conclusions: arrays

47. 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
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, PhD
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