1. Improved effectivity of PGS techniques in clinical IVF: The role of embryo stage and technique selection on efficiency
M. Cristina Magli, Alessandra Pomante, Luca Gianaroli
S.I.S.Me.R. Reproductive Medicine Unit, Bologna, Italy
Istanbul, 1st December 2016

2. Is PGS techniques effective in clinical IVF
Is PGS techniques effective in clinical IVF? The role of embryo stage and technique selection on efficiency
M. Cristina Magli, Alessandra Pomante, Luca Gianaroli
S.I.S.Me.R. Reproductive Medicine Unit, Bologna, Italy
Istanbul, 1st December 2016

3. PGS v.1

4. PGS v.2

5. PGS: RANDOMIZED CLINICAL TRIALS
Higher ongoing pregnancy rate with PGS Molec Cytogenet 2012
Higher sustained Implantation after the transfer of a single euploid blastocyst compared with the transfer of two untested blastocysts
Fertil Steril 2013
Higher Implantation and delivery with PGS
Fertil Steril 2013

6. CCS FOR PGS: RANDOMIZED CLINICAL TRIALS
Dahdouh et al., Fertil Steril 2015

7. CCS FOR PGS: OBSERVATIONAL STUDIES
Dahdouh et al., Fertil Steril 2015

8. Conclusions:
- PGS with the use of CCS technology increases clinical and sustained IRs, thus improving embryo selection, particularly in patients with normal ovarian reserve.
- Results from ongoing RCTs conducted on different patient populations (e.g., decreased ovarian reserve) and different embryo stage biopsy (e.g., PB, day-3) may further clarify the role of this technology.
Dahdouh et al., Fertil Steril 2015

9. PGD IN NON SELECTED PATIENTS
CCS (Comprehensive Chromosome Screening) for 24 chromosomes
RCT on blastocysts
- Good prognosis patients
32 yrs
≥ 2 high quality blastocysts
- Randomization on day 5
Controls: day 5 transfer
PGD: day 6 transfer
- Good prognosis patients
< 35 yrs, at first cycle
no previous miscarriages
Randomization
Assisted hatching on day 3
Transfer on Day 6
Scott et al., Fertil Steril 2013
Yang et al., Molec Cytogenet 2012

10. Shorter time to delivery
LIVE-BIRTH RATE
Shorter time to delivery
21 conventional cycles
31 conventional cycles
n=61
14 deliveries
n=38
13 deliveries
n=56 %
n=20
No. cases

11. CHROMOSOMAL ANALYSIS ON PBs / EMBRYOS
- Biopsy is a damaging procedure
- Biopsies do not predict the embryo chromosome status
Mosaicism
Aneuploidy rescue
- Diagnostic technique not enough accurate (24 chromosomes)
50%
Aneuploidy
40%
30%
20%
Implantation
10%
20-34
35-39
40-45
Maternal age

12. PGS
STAGE OF BIOPSY

13. OOCYTE / EMBRYO BIOPSY Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
Blastocyst biopsy
Polar Body Biopsy
Cleavage stage biopsy
Morula stage biopsy
Trophectoderm
Blastocoelic fluid
Informative only for the maternal counterpart
Moderately predictive of the embryonic status
Mosaicism
Not enough data

14. OOCYTE / EMBRYO BIOPSY Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
Blastocyst biopsy
Polar Body Biopsy
Cleavage stage biopsy
Morula stage biopsy
Trophectoderm
Blastocoelic fluid

15. ANEUPLOIDY TESTING ON DAY 5 EMBRYOS
Pros
Cons – genetics lab
Cons – IVF lab
Paternal, maternal and mitotic contribution to aneuploidy
Short time for diagnosis ( cryopreservation)
Fewer embryos available
More cells available:
more robust diagnosis
Concordance between TE and ICM cells
Increased workload → cryopreservation and thawing
Low level of mosaicism
Concerns over extended embryo culture
Little (if any) impact on embryo – embryonic mass not reduced
Data from the literature
A meta-analysis reports that day TE biopsy has an improved clinical outcome
(Dahdouh et al., 2015)

16. ANEUPLOIDY TESTING ON DAY 5 EMBRYOS
Conclusions
Cleavage stage biopsy + aCGH does not eliminate
the effect of maternal age on implantation
Wells

17. IS BIOPSY A DAMAGING PROCEDURE ?

18. ANEUPLOIDY TESTING ON DAY 3 EMBRYOS
Cleavage-stage biopsy markedly reduced embryonic reproductive potential.
Trophectoderm biopsy had no
measurable impact on implantation
→ may be used safely
Scott et al., 2013

19. BIOPSY PREDICTION OF THE EMBRYO CHROMOSOME STATUS
MOSAICISM

20. Embryonic genome activation
Genome stability dependent on oocyte cytoplasmic transcriptomes
Some genes important for cell division are not expressed until blastocyst stage
MITOTIC ERRORS
MOSAIC EMBRYOS
Genome instability
degradation of the oocyte’s mRNA  decreases fidelity of the cell division.

21. SELECTION AGAINST OR CORRECTION OF ANEUPLOIDY
Three underlying mechanisms
suggested:
self correction via anaphase lag, non-disjunction or chromosome demolition;
cell arrest or apoptosis of aneuploid blastomeres and or embryos;
preferential allocation of diploid/aneuploid blastomeres to embryonic or extra-embryonic tissues
Mantikou et al., 2012

22. MOSAICISM IN BLASTOCYSTS
(66 chromosome errors)
mosaic chromosomal errors observed in 11 (15.7%) but only 2 cases were classified as mosaic diploid/aneuploid (2.9%).
No preferential allocation of abnormal cells between ICM and TE
Capalbo et al., 2013

23. DOUBLE TE BIOPSY 14 blastocysts BF collection Whole embryo analysis
TE1 vs TE2
In 1 case the ploidy condition was confirmed but the number of aneuploid chromosomes was different
In 2 cases the ploidy condition was different

24. MOSAICISM - ANEUPLOIDY RESCUE
The clinical consequences of mosaicism depend on 1) when during development the error occurs, and 2) on the proportion of cells that continue to propagate. At the cleavage-stage embryo, the consequences of chromosomal mosaicism are more severe then if it occurs at later stages.
Incidence of mosaicism
from 15 to 90% at the cleavage stage
from 15 to 30% at the blastocyst stage
from 1 to 2% in prenatal diagnosis
a selection mechanism against mosaicism / correction of aneuploidy in the later stages of development
Taylor et al., 2014

25. TROPHECTODERM BIOPSY
There are no detrimental effects on blastocyst development after TE biopsy
Diagnostic efficiency: 96-98% of diagnosed embryos (depending on the lab)
How many cells can be removed?

26. TROPHECTODERM BIOPSY
n. of cells
*p<0.05
**p<0.01
In embryos with a low TE score the number of biopsied cells negatively influences the IR
Zhang et al., Fertil Steril 2016

27. OOCYTE / EMBRYO BIOPSY Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
Blastocyst biopsy
Polar Body Biopsy
Cleavage stage biopsy
Morula stage biopsy
Trophectoderm
Blastocoelic fluid

29. DETECTION OF DNA IN THE BLASTOCOELIC FLUID
TOT: 206
S.I.S.Me.R. data

30. CONCORDANCE STUDY No more than 3 aneuploidies Complex abnormalities
Polar bodies
Blastomeres
Trophectoderm
Blastocoelic fluid
No more than 3 aneuploidies
Complex abnormalities
Euploidy  pregnancy outcome
Work in progress

31. Blastocoelic fluid (n=105)
CONCORDANCE STUDY
Polar bodies(n=35)
Blastomeres (n=70)
Trophectoderm (n=98*)
Blastocoelic fluid (n=105)
79.4
64.3 %
22.8
16.5
12.9 3
4.1
2 sample failed amplification
5 embryos were transferred
PB Vs BF
BB Vs BF
TE Vs BF

32. Blastocoelic fluid (n=105)
CONCORDANCE STUDY
Polar bodies(n=35)
Blastomeres (n=70)
Trophectoderm (n=98*)
Blastocoelic fluid (n=105)
79.4
64.3
97%
95.9%
85.8% %
22.8
16.5
12.9 3
4.1
2 sample failed amplification
5 embryos were transferred
PB Vs BF
BB Vs BF
TE Vs BF

33. PGS - CONCLUSIONS

34. PGS
DIAGNOSTIC TECHNIQUE

35. NGS TECHNOLOGY
Reads are aligned to a reference with bioinformatic software
Each region is sequenced multiple times
DNA is fragmented
ACCTGATGCTAGCTA
TGGCAACTCGATTAA
TGCATGCTTAGTGC
ACCTGATGCTAGCTAGCTTGGCAACTTGATTAACAGTGCATGCTTAGTGC
Differences between the reference genome and the read sequence are identified

36. NGS TECHNOLOGY: BARCODING
BARCODE are added to each sequence
DNA from different samples can be processed together
Reduction of costs

37. Array-CGH
NGS
Increased (trisomy) or decreased (monosomy) chromosome load is more evident with NGS

38. PGS BASED ON NGS TECHNOLOGY
Parallel analysis of multiple sample with the use of barcodes (up to 96 samples in a single run) → reduced costs
Screening of aneuploidies and simultaneous evaluation of single gene disorders, translocation and abnormalities of the mitochondrial genome from the same biopsy
Detection of mosaicism → more clear
No differentiation between balanced vs normal chromosomes
Cost of NGS instruments

39. NGS: DETECTION OF MOSAICISM
Healthy Babies after Intrauterine Transfer of Mosaic Aneuploid Blastocysts.
Greco E, Minasi MG, Fiorentino F.

40. Euploid embryo (array-CGH) Euploid embryo (array-CGH)
CASE
CONTROL
38 patients
38 patients
FROZEN ET
FROZEN ET
Euploid embryo (array-CGH)
Euploid embryo (array-CGH)
MISCARRIAGE
LIVE BIRTH
POC available in 20 cases
NGS re-analysis
Maxwell et al., 2016

41. RESULTS Triploid embryos not detected by array-CGH
Array-CGH may fail to identify some cases of mosaicism and polyploidy →
undetected abnormalities may contribute to early pregnancy loss
Maxwell et al., 2016

42. Mosaicism detected by NGS not present in POC
RESULTS
Mosaicism detected by NGS not present in POC
small quantity of cells sampled
abnormal or mosaic cells may not form metaphases and undergo proper cell division → they could remain undetected on karyotype analysis
Maxwell et al., 2016

43. MOSAIC EMBRYOS:
“The percentage of chromosomally normal cells within a mosaic embryo or the type of chromosomal abnormality may determine its potential to produce an ongoing pregnancy and live birth. Improved detection of mosaicism among embryos may reduce the risk of early pregnancy failure, but it may also screen out embryos with the potential to produce normal offspring. There is limited evidence to guide practitioners in the use of mosaic embryos.”
“Mosaic cell lines may be sporadically distributed within an embryo, and thus, may be an indicator of reduced live birth potential of an embryo, but not true or future euploidy status.”
“Further studies are needed to determine the types of mosaicism that are capable of producing healthy pregnancies.”
Maxwell et al., 2016

44. Recommendations for the laboratory (if reporting mosaic aneuploidies)
For reliable detection of mosaicism, ideally 5 cells should be biopsied, with as little cell damage as possible. If the biopsy is facilitated using a laser, the identified contact points should be minimal and preferably at cell junctions. Overly aggressive use of the laser may result in cell damage and partial destruction of cellular DNA.
Only a validated NGS platform that can quantitatively measure copy number should be used for measurement of mosaicism in the biopsy sample. Ideally, a NGS methodology that can accurately and reproducibly measure 20% mosaicism in a known sample.
For reporting embryo results, the suggested cut-off point for definition of mosaicism is >20%, so lower levels should be treated as normal (euploid), > 80% abnormal (aneuploid), and the remaining ones between 20-80% mosaic (euploid-aneuploid mosaics).

45. Suggested guidelines to prioritize mosaic embryos for transfer
Embryos showing mosaic euploid/monosomy are preferable to euploid/trisomy, given that monosomic embryos (excepting 45, X) are not viable.
If a decision is made to transfer mosaic embryos trisomic for a single chromosome, one can prioritize selection based on the level of mosaicism and the specific chromosome involved.
a. The preferable transfer category consists of mosaic embryos trisomic for chromosomes 1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 17, 19, 20, 22, X, Y. None of these chromosomes involve the adverse characteristics enumerated below.
b. Embryos mosaic for trisomies that are associated with potential for uniparental disomy (14, 15) are of lesser priority.
c. Embryos mosaic for trisomies that are associated with intrauterine growth retardation (chromosomes 2, 7, 16) are of lesser priority.
d. Embryos mosaic for trisomies capable of liveborn viability (chromosomes 13, 18, 21) are of lowest priority, for obvious reasons.