the embryonic factor: pgt-a & nipgt-a...basic research (plachta lab) multicenter concordance...

Carmen Rubio, PhD

Igenomix Headquarters,

Valencia, Spain

The embryonic factor:

PGT-A & niPGT-A

Why perform PGT-A?

Objectives and Indications of PGT-A

Indications

Advanced maternal age (≥35-38 years)

Prior pregnancy/child with chromosomally abnormality

Multiple implantation failures (≥2 failed IVF)

Recurrent miscarriage (≥2 miscarriages)

Severe male factor infertility (low sperm count)

Good prognosis patients: SET

Improve pregnancy and implantation rates

Decrease miscarriage rates and risk of abnormal offspring

Decrease multiple pregnancies if SET if performed

Decrease time to pregnancy and emotional burden

Cost-efficiency

SET: single embryo transfer

Obje

ctiv

es

How to perform PGT-A?

WGA: whole genome amplification

Sequence up to 96

preimplantation

embryos with Ion 530

Chip

Library prepTemplate prep:

Ion Chef SystemSequencing: S5

system

Analysis: Ion

Reporter software

v5.4 or higher

WGA and

fragment library

construction with Ion

SingleSeq Kit

Isothermal

amplification with

Ion Chef System

Data analysis and storage

with Torrent Suite

Software v5.4 or higher

and Ion Reporter Software

and server

NGS protocol Automated library preparation (Ion Chef, ThermoFisher)

High throughout sequencing (S5, ThermoFisher)

Minimize manual sample handling

Short and cost–effective protocol (12-15 hrs approx.)

NGS: millions of sequences from > 200.000 biopsies

Bioinfoserver

Mosaicism algorithm

Machine learning and

transfer of dataFinal results and report validated by

experienced scientist

Smart PGT-A

Customized diagnosis

algorithm

2)

3)


MOSAICISM: Different number of abnormal cells

After proper validation,

four different categories

were defined:

Mosaicism rate was 5.8%: 3.8% low mosaic degree and 2% high mosaic degree.

The incidence of mosaicism was not different for the 9 diagnostic laboratories, low and

high mosaicism rates were also comparable (low mosaic: 2.4-4.4 and high mosaic: 1-2.6).


What´s new?

Conventional Preimplantation Genetic

Testing for Aneuploidies (PGT-A)

Blastocyst biopsyDay 5-day 6

5-8 trophectoderm cells

NGS

Non-invasive methods

for Aneuploidy Testing (ni PGT-A)

Cell free DNA releasedto the media

NGS

Embryos culturedfrom day 4 -day 6

NGS in Spent Blastocyst Media45, -16, XX

NGS trophectoderm biopsy45, -16, XX

Pilot study TE vs. embryonic cell-free DNA in SBM

Serial Washes

Transfer to 10µl drop on D4

Culture up toD5/D6/D7

Standard culture

D4 embryos Washings D5/D6/D7 biopsy

10µl drop

Cell free DNA

Embryo culture modifications to decrease MCC

Rubio et al., FS in press 2019

Ion Torrent™ Technology (S5)

WGA with conventional and modified protocol

30h

30h

30h

48h

48h

72h

Time in culture:

After 5 minutes

C M

C M

C M

C M

C M

C M

WGA / NGS improvements


Pilot study TE vs. embryonic cell-free DNA in SBM

New NGS protocol: results pilot study

RESULTS DAY 5

TE BIOPSY vs. SBM

(N=27)

vs.

INFORMATIVITY

81.8%

CONCORDANCES

63%

RESULTS DAY 6/7

TE BIOPSY vs. SBM

(N=81)

INFORMATIVITY

100%

CONCORDANCES

84%


New NGS protocol: results on day 6/7

DAY 6 CONCORDANCE: TE BIOPSY vs. SBM

CONCORDANT RESULTS

84%

FALSE NEGATIVES:Aneuploid biopsyEuploid media

2.5%

FALSE POSITIVES:Euploid biopsy

Aneuploid media

8.5%

CONCORDANT RESULTSWith different gender

5%

vs.


NGS results TE vs. SBM

ALL CHROMOSOMES CONCORDANT

NGS results TE vs. SBM

FALSE NEGATIVES & FALSE POSITIVES

Pilot Study: Clinical results


Clinical OutcomeEuploid TE/

Euploid SBMEuploid TE/

Aneuploid SBMTOTAL

Number of transfers 17 12 29

Mean maternal age (SD) 37.5 (2.5) 37.4 (2.3) 37.5 (2.4)

Positive pregnancy test 11 (64.7) 4 (33.3) 15 (51.7)

Biochemical pregnancy loss 2 (18.2) 0 2 (13.3)

Clinical pregnancy rate (%) 9 (52.9) 4 (33.3) 13 (44.8)

Clinical miscarriage 0 2 (50.0) 2 (15.4)

Ongoing implantation rate (%) 9 (52.9) 2 (16.7) 11 (37.9)

Preliminary clinical results in SET

Open questions?

Origin of the DNA? From inner cell mass and trophectoderm cells?

Mechanism? Different embryonic DNA replication? Tubulin bridges?

BASIC RESEARCH (Plachta Lab)

MULTICENTERCONCORDANCE

STUDIES

RCTSTUDY

Can we improve current results by decreasing MCC?

Does culture conditions or embryo quality affect the results?

Could be applied with the aim of improving implantation for every IVF patient?

If this is the case, could transfer priorization be an acceptable alternative?

0

20

40

60

80

100

Center 1 Center 2 Center 3 Center 4

Center 5 Center 6 Center 7 Center 8

VALIDATION: Consistent concordance rates between cfDNA and TE biopsy

Co

nco

rdan

ce

Multicenter Concordance Study (8 centres)

Transfer prioritization according to SBM results

Euploid SBM 98% concordant euploid in TE

Non- Informative 50% euploid in TEChaotic (> 6 aneuploid chr 50% euploid in TE

UNTESTED EMBRYOS

Complex Aneuploid (3-5 chr) 75% aneuploid in TE

Aneuploid (1-2 chr) 95 % aneuploid in TE

the embryonic factor: pgt-a & nipgt-a...basic research (plachta lab) multicenter concordance...

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