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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
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