Disclosure

Declared to be member of Illumina advisory board and to be employed at Reprogenetics

Dagan Wells Institute of Reproductive Sciences Oxford, UK

Predictive value of embryonic mitochondrial DNA: reality or legend

Dagan Wells

United Kingdom

• Mammalian embryos inherit mitochondria & mtDNA from oocyte population

• Metaphase II oocytes contain mitochondria and 50,000-550,000 mtDNA copies

• mtDNA quantity remain stable during first three days of development

May-Panloup et al., 2005 ; St John et al., 2010; Eichenlaub-Ritter et al., 2011

• Eukaryotic mitochondria are main cellular powerhouses

• Generate ATP via Oxidative Phosphorylation (OXPHOS)

• mtDNA replication starts at some point after blastocyst formation

• Mitochondria contain one or more copies of their own genome

Mitochondria in preimplantation development

• Early embryos require adequate energy to progress

• Mitochondrial function and mtDNA gene expression critical

during first few days of life

Aims

• Examine relationships between mtDNA content and

female age

embryo ploidy

viability & implantation potential

Fragouli et al., PLOS Genetics, June 2015; Wells et al., J.Med.Genet, 2014

Mitochondria in preimplantation development

Embryos & Clinics

• Total no. of patients: 240 (average female age 38 years, age range 25-42)

• PGD & PGS for various indications

• TE samples from 387 blastocysts assessed (euploid & aneuploid)

• Blastomeres from 39 euploid cleavage stage embryos assessed

• Participating IVF clinics in Europe & USA

• mtDNA quantification via two different strategies

Methodology

Strategy 1: quantitative real-time PCR

Measurement of mitochondrial sequences

• Cytogenetic assessment via aCGH or Next Generation Sequencing (NGS)

Measurement of nuclear DNA sequences

Strategy 2: Next Generation Sequencing

The effect of female age on mtDNA quantity

• mtDNA quantity changes with advancing age

• Result representative of fertilised oocyte mtDNA

• Cleavage stage: mtDNA quantity decreases with advancing female age (P= 0.01)

• Blastocyst stage: mtDNA quantity increases with advancing female age (P= 0.003)

• Result representative of embryonic mtDNA

mtDNA quantity and blastocyst ploidy

• Elevated mtDNA levels in aneuploid vs. euploid blastocysts (P= 0.025)

• Analysis of different blastocyst cohort via NGS

• Aneuploid blastocysts with higher mtDNA levels vs. euploid (P= 0.006)

• mtDNA quantity associated with embryo ploidy

• mtDNA quantity relationship with embryo ploidy is independent of female age

• Embryos establishing viable clinical pregnancies contained significantly lower mtDNA levels (P= 0.0066) compared with those not implanting

Total no. of patients with ETs: 87 (average female age: 38.3 years)

mtDNA quantity and blastocyst implantation ability

• mtDNA levels were predictive of a euploid blastocyst’s implantation ability

Total no. of embryos transferred: 92 (83 SETs & 4 double ETs)

Total no. of embryos leading to ongoing pregnancies: 44

Total no. of embryos which did not implant: 48

28% of euploid blastocysts that fail

to implant have elevated mtDNA

Normal mtDNA levels

Elevated mtDNA levels

Pregnant Not-pregnant

Retrospective analysis

mtDNA quantity

92 euploid blastocysts

Known outcome

Fragouli et al., 2015 PLoS Genetics

0

0,005

0,01

0,015

0,02

• Data analysis led to establishment of mtDNA quantity implantation threshold

• All of the embryos with mtDNA quantities above threshold failed to implant

• ~30% of non-implanting euploid embryos contain high levels of mtDNA

• 100% of implanting embryos contain amounts of mtDNA below the threshold

• No association with blastocyst morphology

mtDNA quantity and blastocyst implantation ability

Timing of mtDNA replication in the human embryo

• When do elevated mtDNA levels first arise?

• mtDNA quantification of blastomeres from 39 euploid cleavage stage embryos

• Clinical details: 27 SET & 7 DETs

17 singleton pregnancies (from SETs) & 1 set of twins (from DET)

20 failed implantations

• No significant difference in mtDNA quantities of implanting & non- implanting embryos (P= 0.7)

• mtDNA replication starts after blastocyst formation

• Elevated mtDNA levels first arise after first embryonic differentiation

Blinded prospective prediction of IVF outcome

• Evaluate the accuracy & efficiency of mtDNA implantation threshold

• Corresponding embryos transferred in 66 SETs & 6 DETs

• 37 ongoing pregnancies & 39 failed implantations

• Blinded prospective mtDNA quantification of 78 TE samples

(average female age 37 years, 8 IVF clinics)

• All embryos with mtDNA quantities above threshold failed to implant

Egg mitochondria. As old as the egg Embryonic mitochondria (created after genome activation)

Fragouli et al. (2015) PLOS Genetics, Fragouli et al. (2015) SART prize

Normal Blastocyst mtDNA level = high implantation

Elevated Blastocyst mtDNA level = low implantation

Diez-Juan et al (2015) Fertil Steril

Egg mitochondria Mitochondria of embryonic origin

Cell with high mtDNA biopsied

Biopsy causes mtDNA depletion* Embryo arrests and Test classifies it as non-viable (yet actually could have been viable)

Biopsy non detrimental. Embryo classified As viable.

* Van Blerkom et al., 2000; Katayama et al., 2006; El-Shourbagy et al., 2006; Murakoshi et al. 2013

Pregnant Not-pregnant 0

0,005

0,01

0,015

0,02

Data obtained using the MitoGrade test

Blinded prospective study

Normal mtDNA levels

Elevated mtDNA levels

40% of euploid

blastocysts that fail to implant have elevated

mtDNA

16% of all euploid blastocysts have elevated mtDNA

Single embryo transfer pregnancy rates

>75%

Pregnancy loss rate ~5%

(mean age 37)

Euploid implanting (40%)

Aneuploid (35%)

Euploid not implanting - Unknown reason (25%)

no PGS selection: 40% implantation

PGS selection: 62% implantation

Example: patient 35 year old, blastocyst transfer

Embryo selection – Using PGS to find viable embryos

Aneuploid (35%)

Euploid not implanting - Unknown reason

Euploid implanting (40%)

Euploid not implanting - Elevated mtDNA PGS selection: 62% implantation

PGS + MitoGrade selection: 70% implantation (estimated)

Example: patient 35 year old, blastocyst transfer

Embryo selection – Finding viable embryos

Conclusions

• Threshold of mtDNA content established, above which implantation for a euploid blastocyst does not occur

• Transfer of euploid embryo does not guarantee implantation

• Large nonselection study Fragouli (ESHRE, 4.15pm Hall 1), paired transfer study

• Association between mtDNA quantity, aneuploidy risk, female reproductive aging

• Elevated mtDNA quantities arise after blastocyst differentiation

• Elevated mtDNA quantities characteristic of embryos under stress ?

• Findings consistent with the ‘quiet embryo’ hypothesis ?

Fragouli et al., PLOS Genetics, June 2015

• Confirmatory data Spinella (ESHRE, 4pm Hall 1) – Next Generation Sequencing

Acknowledgements University of Oxford & Reprogenetics UK:

Dagan Wells Samer Alfarawati Katharina Spath

dagan@reprogenetics.co.uk

Reprogenetics: Santiago Munne Jacques Cohen

Krithika Ravichandran Emmeline Liu

Michalis Konstantinidis Lia Rubistello

Pere Colls

Reprogenetics Spain: Mireia Sandalinas

Carles Giménez

Illumina: Fiona Kaper

Claude-Edouard Michel Andrew Craig

Felix Kokocinski

Collaborating IVF clinics

Tecnobios Procreazione: Andrea Borini

Nicoletta Tarozzi

NYU Langone Medical Center: Jamie Grifo

Caroline Mccafrey David McCulloh