1. Preimplantation Genetic Screening for IVF: Any Benefit for Baby Take Home rate
Eray ÇALIŞKAN MD
Obstetrics and Gynecology
Bahçeşehir University School Of Medicine, Turkey
«scientia et amore vitae»

2. Definition Preimplantation Genetic Diagnosis
To search for genetic disease known to be present in one or both of the parents
Preimplantation Genetic Screening
To screen the embryo for the presence of uneuploidy where the parents are known to be genetically normal

3. Laser Q PCR aCGH Blastocyst NGS Embryo Biopsy Technique Chemical
Mechanical
FISH-PCR
Q PCR
aCGH
NGS
Polar body
Blastomere
Blastocyst
Staff Experience
Laboratory Quality
Implantation
On going
Live birth
Single ET
Double ET
Embryo Transfer
Mosaicism
Pregnancy
Day 5
Day 6
Day 7?
Fresh ET
Frozen ET
-All freeze
Per cycle
Per transfer

4. PGS TECHNICAL PROBLEMS
Failure to obtain genetic material
Nuclear abnormalities
Multinucleation
Binnucleation
Apoptotic
Anuclear
The single cell may not represent the embryo
Mozaicism 20%

5. Rationale for Preimplantation Genetic screening?
The reason for implantation failure is uneuploidy in 50% of the cases
70% of uneuploidies occur during the first meiotic division of the oocytes
Blastocysts with good morphology have 30% uneuploidy rate
Munne et al., RBM online, 2003

6. PGS 1.0
FISH based PGS

7. FISH BASED EXPERIENCE
RCT revealed that PGS did not increase baby take home rates
Staessen et al 2004, 2008; Mastenbroek 2007; Hardarson 2008; Meyer et al 2008; Debrock 2009)
BUT
FISH first generation technology
8 chromosome FISH – worst scenario FNR 27%
Embryo biopsy experience of the centers ?
The timing: polar body, day 3 and blastocyst
Johnson et al, Hum Reprod, 2010

8. FALSE POSITIVE RATE IN FISH TECHNOLOGY
When FISH diagnosis was monosomy 65% of embriyos were euploid with array CGH
When FISH diagnosis was trisomy 47% of the embryos were found to be euploid by array CGH
When FISH diagnosis was complex uneuploidy 63% of the embryos were euploid by array CGH
Northrop et al, Hum Reprod, 2010

9. FISH technology Array CGH
Allel drop out per probe in FISH technology 2-3%
6 probe False diagnosis up to %
8 probe False diagnosis up to 16-24%
False diagnosis in all 23 chromosomes 2%
Random loss of one allel
Over amplification
Under amplification
Handyside 2004, Rendwick 2006, Spits 2006, Glentis 2009

10. Spontaneous Abortion Data
Bulgu
FREKANS
45,X 18
Triploidi 17
Trisomi 16
16.4
Trisomi 22
5.7
Trisomi 21
4.7
Trisomi 15
4.2
Trisomi 13
3.7
Trisomi 18 3
Diğer trizomiler
14.3
Tetraploidi 6
Dengesiz translokasyonlar
Diğer 4
51%

11. PREIMPLANTATION GENETIC SCREENING
Was aimed to diagnose most common chromosomal abnormalities found in spontaneous abortions
Triploidi
Monosomi X
Trizomi 16
Trizomi 22
Tetraploidi
Trizomi 15
Trizomi 21
Common chromosomes screened with FISH
13,18,21, X, Y – 16, 22
PPV 51% - 16% FNR

12. VIABILITY AND IMPLANTATION
Abortion data and viable uneuploidies do not provide enough information about in vivo embryos, some uneuploidies can implant BUT
We were unaware of never implanting chromosomal aberrations?
We had the dogma that several chromosomes have a tendency for uneuploidy?

13. 557 embryo test – GENETİKON-Arrayland
8 probe FISH
45% Detection Rate
Drop-out %16-24

14. PB 1
Blastocysr ICM 1

15. PGS 2.0
Array CGH
Next Generation Sequencing

16. Euploid Trophoectoderm

17. NGS vs a CGH 100% concordance Similar pregnancy
Yang et al, BMC Medical Genomics, 2015

18. qPCR EUROFERTİL® Flourescent carrying DNA primers
Flourescents detected and analyed by computerized systems
EUROFERTİL®

19. 384 well plates are used
Chromosomal copy numers are calculated
Decision for euploid/uneuploid using chromosomal copy numbers

20. Concordance between ICM and trophectoderm
Concordance for euploidy confirmed by CGH or aCGH (97-100% concordance).
Fragouli et al., Hum Reprod, 2008
Capalbo et al., Hum Reprod, 2013
- Uneuploid cells are more common in the trophectoderm
Gleicer and Orvieto J Ovarian Res 2017
-Uneuploid trophoectoderm and euploid inner cell mass discrepency is up to 50%
Gleicher et al RBE 2016

21. MOSAICISM Sequential trophoectoderm and inner cell mass biopsy
42.3% all cells are euploid
30% all cells are uneuploid
32.4% si mosaic
15.4% mixed uneuploidy from different cell lines,
17% both euploid and uneuploid cells together
Mosaic diploid– 6% of the total embryos (20% of uneuploid blastocysts have euploid inner cell mass)

22. Mosaicism Trophoectoderm biopsy
18 mosaic embryo transfer 6 euploid delivery
5 single uneuploid transfer 3 euploid embryo
6 Uneuploid dx first than, euploid in another lab test with array CGH, ET and 5 euploid baby one biochemical pregnancy
All these questions the true diagnosis of uneuploidy
Gleicher Fertil Steril 2015, Greco Curr Genom 2012, Gleicher RBE 2016

23. Abnormalities reaching blastocyst stage
Arrested Arrested reached
day 3 day 4 blastocyst
Euploid:
Total 9 (28%) 2 (6%) 21 (66%)
Aneuploid:
monosomies (9%)
trisomies (39%)
and mosaics (>38% abn.) (0%)
Total 48 (58%) 19 (23%) 16 (19%)
Other mosaics:
2n/4n (>38% abnormal) (33%)
2n/aneuploid or chaotic (11%)
Total 57 (70%) 15 (18%) 11 (13%)
Polyploid: 11 (79%) (21%)
Haploid: 4 (80%) 1 (20%) (0%)
Sandainas et al, Hum Reprod, 2001

24. Advanced Emryo Cultures: More Uneuploids Reach Blastocyst Stage
trisomy
Monosomy
complex
mosaic
Northrop et al 2010 6 5 1 12
Fragouli et al 2011 7 2 14
Colls et al. 2013 16 10
% of total abnormal
31%
24%
10%
35%
Sandalinas et al. 2010
46% 7%
10%
36%
Fragouli et al. Hum. Reprod. 2011, Northrop et al, Mol Hum Reprod. 2010, Colls et al. ASRM, 2013, Sandalinas et al., Hum Reprod, 2010

25. Morphological Evaluation of Embryos and array CGH results
Embryo Grade
Euploid
1.5
Mosaic Eploid
1.9
Chaotic Euploid
1.7
Uneuploid
2.2
Segmental Errors
1.4
Johnson et al, Hum Reprod, 2010

26. TÜMGEN® and EUROFERTİL ®
Grade I uneuploid ⁺ 11 ⁺ 15 ⁺ 20
Grade I uneuploid ⁺ 11 ⁺ 15 ⁺ 20
Male 47 XYY, female 46 XX
TÜMGEN® and EUROFERTİL ®

27. TÜMGEN® and EUROFERTİL ®
+ 2, +5, +12, - 13, +18
Euploid
Mixed Uneuploid
-2, -11p
TÜMGEN® and EUROFERTİL ®

28. TÜMGEN® and EUROFERTİL ®
Euploid
-1,+5,+6,+12,-13,-14,+18
TÜMGEN® and EUROFERTİL ®

29. Effect of D 3 vs Trophoectoderm biopsy
cleavage stage blastocyst
biopsy not biopsy not
Implantation rate 31% 53% 52% 54%
P< N.S.
Blastocyst biopsy, more DNA-less no results, less mosaicism, reduced impact of embryo biopsy, less embryos to process, facltates single embryo transfer, uterine environment optimized after frozen cycle. Disadvantages: not all embryos reach blastocyst stage
Treff et al., Fertil. Steril. 2011

30. No results day 3 vs Trophoectoderm biopsy
Biopsy Embryos Centers*
stage undiagnosed range
Cleavage % 1% - 5%
Blastocyst % 0% - 18%
Most experienced
Untrained
Munne S, 2013, ASRM

31. Euploidy decreased with age but not with cohort size
# of blastocysts
% normal embryos
egg donors
< years
years
years
years
>42 years
1-3
65%
63%
50%
40%
22%
14%
4-6
60%
61%
52%
39%
27%
17%
7-10
64%
62%
53%
25%
16%
>7-10
56%
43%
29%
N = 3,571 cycles and 19,356 embryos, up to 8/2013.
Ata, et al. Reprod Biomed Online, 2012

32. % of patients with normal embryos
Percent of Women with Euploid Embryos: ET possible
# of blastocysts
% of patients with normal embryos
egg donors
< years
years
years
years
>42 years
1-3
86%
85%
72%
60%
58%
24%
4-6
95%
97%
88%
69%
54%
7-10
100%
99%
96%
92%
65%
>7-10
98%
83%
N = 3,571 cycles and 19,356 embryos, up to 8/2013.
Ata, et al. Reprod Biomed Online, 2012

33. Cycles with no Euploid Embryos
Harton, Munne et al., Fertil Steril, 2013

34. On Going Pregnancy rate / ET
Harton, Munne et al., Fertil Steril, 2013

35. Overall Pregnancy Rate*
Harton, Munne et al., Fertil Steril, 2013

36. CGH and frozen transfer
Cycles Mat. Prev. embryos implant.
age failed replaced (+ sac) cycles
CGH : %
control : %
p=0.0003
Schoolcraft et al., Fertil. Steril., 2010

37. aCGH + fresh transfer, <35 years old
Control PGD (aCGH)
Patients 48 55
Maternal age <35 <35
Biopsy on day 5 no yes
Transfer on day 6 day 6
Embryos euploid (N) n/a 53.2% (425)
Embryos replaced (av.) 48 (1) 55 (1)
Pregnancy rate (sac) 45.8% 70.9% p<0.05
Ongoing preg rate 41.7% 69.1% p<0.05
Multiple pregnancies 0 0
Yang et al. Molec Reprod, 2012

38. qPCR + fresh transfer PGS Control age 32.2 N 72 83 blastocysts 8 7.9
Good prognosis patients (average 8 blastocysts)
Control replaced on day 5, test biopsied on day 5 and replaced on day 6
PGS
Control
age
32.2 N 72 83
blastocysts 8
7.9
Emb replaced
1.9
2.0
implantation
79.8%
63.2%
P=0.002
Sustained implant
66.4%
47.9%
P=0.03
Delivery rate
84.7%
67.5%
P=0.01
Scott et al., 2013 Fertil Steril.

39. BEST trial: One tested euploid vs. Two untested
Delivery Rate
1 euploid blastocyst
2 untested blastocyst
Initial Fresh and Frozen transfer
60.6%
65.1% NS
Twin
30%
Frozen transfer
41.1%
46.1%
6.2%
23%
Forman et al., Am J Obstet Gynecol, 2014

40. PGS for Idiopathic Recurrent Pregnancy Loss
PGS decrease miscarriage in women with idiopathic previous recurrent pregnancy loss
Werlin et al, Fertil Steril, 2003, Munne et al, Fertil Steril 2005, Garrisi et al, Fertil Steril 2009, Rubio et al, RBM Online 2009, Hodes-Wertz et al, Fertil Steril 2012

41. Results of PGS by aCGH for RPL: biopsy stage effect
day preg. % loss % loss
biopsy cycles expected after PGS
Day % 9% p<0.001
Day % 3% p<0.001
Total % 6% p<0.001
Hodes-Wertz et al., Fertil Steril, 2012

42. What do meta-analysis tell us?

43. Implantation
Chen et al, PLOSone, 2015

44. RCT
Dahdough et al, Fertil Steril, 2015

45. Observational Studies
Dahdough et al, Fertil Steril, 2015

46. Clinical Pregnancy Rate
Chen et al, PLOSone, 2015

47. Ongoing Pregnancy Rate
Chen et al, PLOSone, 2015

48. Live Birth Rate
Chen et al, PLOSone, 2015

49. Miscarriage Rate
Chen et al, PLOSone, 2015

50. Multiple Pregnancy Rate
Chen et al, PLOSone, 2015

51. Women aged 40-43
Lee et al, J Asist Reprod Genet, 2015

52. Recently Challenged Ideas
Most IVF cycles fail because of aneuploid embryos
Their elimination prior to embryo transfer will improve IVF outcomes
A single trophectoderm biopsy (TEB) at blastocyst stage is representative of the whole TE
TE ploidy reliably represents the inner cell mass (ICM)
Ploidy does not change (i.e., self-correct) downstream from blastocyst stage

53. CONCLUSION
Mosaicism may decrease the chance of embryo transfer in women with few embryos after PGS
PGS significantly improves ART outomes per embryo transfer not improves per started cycle
PGS eliminates the effect of maternal age on implantation
Poor Responder Women reported to have no uneuploid embryo for transfer can have second biopsy or transfer