The embry bryonic onic facto ctor: PGT PGT-A A & niPGT - - PowerPoint PPT Presentation

Carmen Rubio, PhD Igenomix Headquarters, Valencia, Spain

The embry bryonic

• nic facto

ctor: PGT PGT-A A & niPGT GT-A

Why y perf rform

• rm PGT-A?

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

Objectives

How to perf rform

• rm PGT-A?

A?

WGA: whole genome amplification

Sequence up to 96 preimplantation embryos with Ion 530 Chip

Library prep Template prep: Ion Chef System Sequencing: 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 data Final results and report validated by experienced scientist

Smart PGT-A Customized diagnosis algorithm 2) 3)

How to perf rform

• rm PGT-A?

A?

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).

How to perf rform

• rm PGT-A?

A?

What´s s new? w?

Conv nven ention

• nal Preimp

eimplanta ntati tion

• n Gene

neti tic Testi ting ng for Aneu euploi

• idies

es (PGT-A) A)

Blastocyst biopsy Day 5-day 6 5-8 trophectoderm cells

NGS

Non-in invas asive ive meth thods ds for Aneup uploidy Testi ting (ni ni PGT-A) A)

Cell free DNA released to the media

NGS

Embryos cultured from day 4 -day 6

NGS in Spent Blastocyst Media

45, -16, XX

NGS trophectoderm biopsy

45, -16, XX

Pilo lot study dy TE vs. embry bryonic

• nic cell-fr

free DNA in SBM

Serial Washes Transfer to 10µl drop on D4 Culture up to D5/D6/D7 Standard culture

D4 embryos Washings D5/D6/D7 biopsy 10µl drop Cell free DNA

Embryo ryo culture re modifi difica cati tions s to decrea rease se MCC

Rubio et al., FS in press 2019

Ion Torrent™ Technology (S5)

WGA with conventional and modified protocol

30 h 30 h 30 h 48 h 48 h 72 h Time in culture: After 5 minutes C M C M C M C M C M C M

WGA / N NGS improvemen ements ts

Rubio et al., FS in press 2019

Pilo lot study dy TE vs. embry bryonic

• nic cell-fr

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%

Rubio et al., FS in press 2019

New NGS protocol: results on day 6/7 DAY 6 CONCORDANCE: TE BIOPSY vs. SBM

CONCORDANT RESULTS

84%

FALSE NEGATIVES: Aneuploid biopsy Euploid media

2.5%

FALSE POSITIVES: Euploid biopsy Aneuploid media

8.5%

CONCORDANT RESULTS With different gender

5% vs.

Rubio et al., FS in press 2019

NGS results TE vs. SBM

ALL CHROMOSOMES CONCORDANT

NGS results TE vs. SBM

FALSE NEGATIVES & FALSE POSITIVES

Pilot Study: Clinical results

Rubio et al., FS in press 2019

Clinical Outcome Euploid TE/ Euploid SBM Euploid TE/ Aneuploid SBM TOTAL 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) 2 (13.3) Clinical pregnancy rate (%) 9 (52.9) 4 (33.3) 13 (44.8) Clinical miscarriage 2 (50.0) 2 (15.4) Ongoing implantation rate (%) 9 (52.9) 2 (16.7) 11 (37.9)

Prelimin iminary ary clinica ical result lts s 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) MULTICENTER CONCORDANCE STUDIES RCT STUDY

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?

20 40 60 80 100 Center 1 Center 2 Center 3 Center 4 Center 5 Center 6 Center 7 Center 8

VALI LIDATIO ATION: N: Cons nsiste stent nt conc ncor

• rdanc

nce rates tes betwe tween en cfDNA NA and TE biopsy

• psy

Concordanc e

Multicenter Concordance Study (8 centres)

Transfer prioritization according to SBM results Euploid SBM  98% concordant euploid in TE Non- Informative  50% euploid in TE Chaotic (> 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