IGENOMIX and Valencian Infertility Institute Foundation ... · IGENOMIX and Valencian Infertility Institute Foundation (FIVI)/INCLIVA Valencia, ... NGS vs. arrayCGH: ... For whole-chr

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Carmen Rubio IGENOMIX and Valencian Infertility Institute Foundation (FIVI)/INCLIVA Valencia, Spain

Declared no potential conflict of interest.

www.excemed.org

IMPROVING THE PATIENT’S LIFE THROUGH

MEDICAL EDUCATION

Preimplantation Genetic Screening

Improves clinical outcomes

24-25 September 2015

Madrid and Alicante, Spain

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Only morphological criteria

fails selecting the best

embryo.

Transference of “good

morphology” embryos not

always means “good quality”

embryos.

Yang et al., 2012

Preimplantation Genetic Screening (PGS)

Indications

Advanced maternal age (≥38 years)

Prior pregnancy /child chromosomally abnormal

Multiple implantation failures (≥2 failed IVF)

Recurrent miscarriage (≥2 miscarriages)

Severe male factor (low sperm count)

Improve implantation

Decrease miscarriage rates

Decrease risk of abnormal offspring

Assesment of embryo viability:


2014

2008 2010 2012

1991-1995

FISH Array

Comparative

Genomic

Hybridization

(aCGH)

Single-nucleotide

polymorphism

(SNP) microarray

Quantitative

polymerase

chain reaction

(qPCR)

Next-Generation

Sequencing

(NGS)

2-12 chromosomes 24 chromosomes

PCR

Analysis of a identified single gene disease 24 chromosomes

+ Mito DNA; mutations

Preimplantation Genetic Screening: HOW


Cy3 Cy5

24sure

BlueGnome

2684 clones

1Mb coverage

BlueFuse Multi

software

Biopsy

Cell (s) loading

Amplification (~ 3 hrs)

(98.3%)

Labelling (2 hrs)

DNA precipitation (~ 1 hrs)

Hybridisation (5–12 hrs)

Washing (~ 1/2 hr)

Scanning

Sample

1

Sample

2

Array CGH Work-flow


Partial gain chromosome 2p

Euploid embryo

Complex aneuploidy

Chaotic pattern

Loss of chromosome 13

Gain of chromosome 21

Array CGH profiles


Next Generation Sequencing (NGS) work-flow

Tagmentation DNA library

Complementary nucleotide sequencing Matching with Reference DNA

DNA

amplification

Lysis

Sequence length: 35 nucleotides per read. Number of reads:

≥1x106


Concordance rate between aCGH and NGS: 100 %

arrayCGH

NGS

NGS vs. arrayCGH: whole-chromosome aneuploidies (2 studies)

N = 47 (Vera et al., 2014 ESHRE) N = 55 (Fiorentino, et al. 2014 FS)

Illumina platform

24sure platform (BlueGnome-

Illumina)


Clinical NGS data

PGS cycles 55

Female age 39.9 (SD2.4)

ET 47 (85.5%)

PR/ET 30 (63.8%)

IR 64%

Similar clinical data NGS vs arrayCGH

(Fiorentino, et al. 2014 FS)

AMA patients

NGS1 SNParray1 arrayCGH2

PGS cycles 114 292 55

Female age 33.6 31.4 31.2

ET 80 (70.2%) 203 (69.5%) 55 (100%)

PR/ET 49 (61.2%) 115 (56.7%) 39 (70.9%)

OPR/ET 42 (52.5%) 98 (48.3%) 38 (69.1%)

IR 52.6% 47.6% 70.9%

Miscarriage 14.3% (7/49) 14.8% (17/115) 2.6% (1/39)

1(Tan et al., 2014 GigaScience); 2(Yang et al., 2012 Molec Cytog)

Young good prognosis

patients


Similar clinical data NGS vs arrayCGH


Where are we? Where we go?

For blastomere and trophectoderm samples

For whole-chr and segmental aneuploidies

Validated technology

Mitochondrial DNA analysis

Embryo viability markers

Single gene disorders

Personalized Reproductive

Medicine

NGS


*p = 0.0012

**p = 0.027

Day-3 biopsies Trophectoderm

Mitocondrial DNA cuantification

(Diez et al., FS 2015)

N= N=

Preimplantation Genetic Screening: WHEN

24-chromosome screening (CCS)

Day-3 biopsy

Day-5 /Day-6 transfer

Blastocyst biopsy


VS.

Day-3 Blastocyst

No. of cycles 4118 197

Mean age 39.7 37.7

Mean MII 9.5 13.2

Mean analyzed 6.2 5.4

% abnormal embryos 81.2 64.0*

% chaotic pattern 16.2 2.0*

% complex aneuploidy 19.3 6.9*

% partial aneuploidies 5.3 6.5

N=5,115 cycles

(25,675 embryos)

(*p<0.05)


4918 cycles with day-3 biopsies

197 cycles with day-5/6 biopsies

2011-2014 percentage of abnormal embryos (5115 cycles)


arrayCGH on day-3 biopsy

49/50 blastocyst confirmed

FPR = 2%

Vs.

Day-3 Biopsy Day-5 Biopsy

Vs.

Low false positive rate in both types of biopsies

57/59 blastocyst confirmed

FPR = 3.4%

(Mir, et al., 2015)

arrayCGH on whole blastocyst


VS.

Day-3 (< 38 yrs) Blastocyst (< 38 yrs)

No. of cycles 1184 98

Mean age 34.6 33.8

Mean MII 11.9 13.8

Mean No. analyzed 6.6 6.0

Embryo transfers 81.3 87.7

PR/transfer 60.0 60.5

Implantation rate 50.0 55.0

PR/cycle 49.0 53.1

Miscarriage rate 11.3 13.8

N=1,282 cycles

(NS)

Preimplantation Genetic Screening: TO WHOM

(NS)

RM <38yrs RIF <38yrs MF <38yrs PTP <38yrs AMA ≥38

Mean Age 34.6 34.9 34.1 35.1 41.0

Abnormal embryos 68.3 66.5 66.1 68.9 86.3

Embryo transfers 75.2 79.9 82.9 85.0 37.9

PR/transfer 57.3 56.7 63.2 61.8 51.1

Implantation rate 46.3 45.6 54.1 59.2 46.2

PR/cycle 43.1 45.2 52.4 52.5 19.4

Miscarriage rate 13.8 10.0 11.6 9.5 9.9


0

10

20

30

40

50

60

70

80

90

100

38 (n=267) 39 (n=378) 40 (n=634) 41 (n=732) 42 (n=489) 43 (n=303) 44 (n=171) 45-46 (n=131)

% Abnormal % Transfer PR/transfer IR PR/cycle

Results according to maternal age


Day-0 Day-1

Polar Body

biopsy

Blastocyst

biopsy

Cleavage stage

biopsy

Advanced maternal age (38 - 41

years)

Severe male factor (≤ 2 million/mL)

Study groups

ClinicalTrials.gov NCT01571076


* p<0.05 T-Student and Fishers´ test; 12 weeks ongoing pregnancies

VS. Blastocyst PGS Blastocyst PGS

No. of cycles performed 86 75 41 44

Mean age (SD) 39.0 (2.8) 39.5 (3.0) 32.6 (3.4) 33.0 (2.8)

Percentage of transfers 96.5 70.7 97.6 88.6

Mean embryos/transfer (SD) 1.8 (0.6) 1.3 (0.7) 1.9 (0.6) 1.5 (0.7)

No. of miscarriages (%) 17 (43.6) 1 (3.3) 6 (26.1) 1 (3.4)

Ongoing PR/transfer 26.5 60.4 42.5 71.8

Ongoing PR/cycle 25.6 42.7 41.5 63.6

Ongoing IR 18.4 58.6 22.7 55.2

*

*

* * *

*

*

* * *

< 38 years ; < 2 mill spz/mL 38-41 years ; > 5MII


RCT AMA


RCT MF VS.

(May 2012-April 2014)

Rubio et al., ESHRE and ASRM 2014


Women < 35 years

First IVF attemp

No previous miscarriages

(Yang et al., 2012)

RCT, blastocyst biopsy with single embryo transfer (SET)


Women 21-42 years

First IVF attempt

No previous miscarriages

(Scott et al., FS 2013)

Women <43 years

AMH ≥1.2 ng/ml

FSH <12 IU/L

(Forman et al., FS 2013)

Single embryo transfer (SET)


PGD

PGS

Mandatory Day-5,6 biopsy:

PGS and PGD analysis in the same sample

- Monogenic disease

+ - Advanced maternal age

- Recurrent miscarriage

- Repetitive implantation failure

- Severe male factor

- Previous pregnancy with trisomy

- Abnormal karyotype (X0,XXX,XXY,XYY)

- Translocations/inversions analyzed by aCGH

♀≥ 38 years

PGD PGD + PGS

OPR/T

42,9%

50% of NORMAL-CARRIER embryos

for monogenic diseases are carriers

of chromosomal abnormalities

OPR/T

33,5%

Combined PGD+PGS

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Conclusions

24-chromosome analysis by Array-CGH NGS mitDNA

PGS: How ?

PGS: When ?

To whom ?

High aneuploidy risk: AMA, RM, RIF, MF, PTP

Young good prognosis patients

Single embryo transfer

PGD +PGS

Day-3 or trophectoderm biopsies offer similar results in

similar good prognosis population.

For AMA and low ovarian response day-3 could be

more informative.

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Valencia-Spain

Miami-USA

Sao Paulo-Brasil

New Delhi-India

Dubai-UAE

Documents

IGENOMIX and Valencian Infertility Institute Foundation ... · IGENOMIX and Valencian Infertility Institute Foundation (FIVI)/INCLIVA Valencia, ... NGS vs. arrayCGH: ... For whole-chr