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USA | Europe | Asia | South America
Embryo Testing
Reprogenetics Inside
Products of Conception Analysis (POC)
Paternal Genetic Testing
Genetic Counseling
Embryology and Biopsy Services
Educational Support Services
LEADER IN PGD
COMPREHENSIVE
REPRODUCTIVE
GENETIC ANALYSIS
Mitochondrial DNA levels and aneuploidy
a new system to assess implantation potential
Jacques Cohen(Reprogenetics)
SWES, 2016
USA | Europe | Asia | South America
Embryo Testing
Reprogenetics Inside
Products of Conception Analysis (POC)
Paternal Genetic Testing
Genetic Counseling
Embryology and Biopsy Services
Educational Support Services
LEADER IN PGD
COMPREHENSIVE
REPRODUCTIVE
GENETIC ANALYSIS
Disclosures and Affiliations
Consultant and Founder of Reprogenetics
Director ART Institute of Washington
Product Developer and Founder of Life Global
Product Developer and Shareholder Althea Science
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Embryo Testing
Reprogenetics Inside
Products of Conception Analysis (POC)
Paternal Genetic Testing
Genetic Counseling
Embryology and Biopsy Services
Educational Support Services
LEADER IN PGD
COMPREHENSIVE
REPRODUCTIVE
GENETIC ANALYSIS
Points to cover
Is embryo selection desired?
Hype and hype cycle
Quantifying mtDNA (mitograde)
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Embryo Testing
Reprogenetics Inside
Products of Conception Analysis (POC)
Paternal Genetic Testing
Genetic Counseling
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Educational Support Services
LEADER IN PGD
COMPREHENSIVE
REPRODUCTIVE
GENETIC ANALYSIS
Embryo Selection MethodsStatic oocyte and embryo morphology (Trounson and Wood, 1981; Edwards et al, 1984)
Follicular fluid marker (Botero-Ruiz et al, 1984; Gazvani et al, 2002; Lédée et al, 2008)
Cumulus cell marker (McKenzie et al, 2004; Zhang et al, 2005; Anderson et al, 2009;
Assou et al, 2010)
Sperm selection methods (Huszar et al, 2007; Bartoov et al, 2002)
PGS, mitochondria, de novo mutations (Munné et al, 1995; Fragouli et al, 2015;
Peters et al, 2015)
Time-lapse – single markers, algorithms, automation (Payne et al, 1997;
Lemmen et al, 2008; Pribenszky et al, 2010; Wong et al, 2010; Meseguer et al, 2011)
Omics (Katz-Jaffe and McReynolds, 2013; Cortezzi et al, 2011; Botros et al, 2008;
Nel-Themaat and Nagy, 2011)
Non-coding RNA (McCallie et al, 2010)
Biochemical markers (Fishel et al, 1984; Conaghan et al, 1993; Botros et al, 2008)
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Embryo Testing
Reprogenetics Inside
Products of Conception Analysis (POC)
Paternal Genetic Testing
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LEADER IN PGD
COMPREHENSIVE
REPRODUCTIVE
GENETIC ANALYSIS
Is Embryo Selection Desired?
USA | Europe | Asia | South America
Embryo Testing
Reprogenetics Inside
Products of Conception Analysis (POC)
Paternal Genetic Testing
Genetic Counseling
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LEADER IN PGD
COMPREHENSIVE
REPRODUCTIVE
GENETIC ANALYSIS
Technology Lovers - Banksy
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Embryo Testing
Reprogenetics Inside
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LEADER IN PGD
COMPREHENSIVE
REPRODUCTIVE
GENETIC ANALYSIS
USA | Europe | Asia | South America
Embryo Testing
Reprogenetics Inside
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GENETIC ANALYSIS
20 + 20 + 20 + 20 + 20 = 100
10 + 10 + 10 + 10 + 10 +10 + 10 + 10 + 10 + 10 = 100
The endpoint is the same!
The second path takes longer and is more costly, medically complicated and painful!
USA | Europe | Asia | South America
Embryo Testing
Reprogenetics Inside
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LEADER IN PGD
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GENETIC ANALYSIS
Introducing new technology, such as time-lapse, PGS 2.0 and qt mtDNA
(mitograde)
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Embryo Testing
Reprogenetics Inside
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COMPREHENSIVE
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GENETIC ANALYSIS
USA | Europe | Asia | South America
Embryo Testing
Reprogenetics Inside
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LEADER IN PGD
COMPREHENSIVE
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GENETIC ANALYSIS
Gartner – Palmer* Hype Cycle
11
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GENETIC ANALYSIS
Advantages of time-lapse
Better selection and improved implantation?
Quality Management Tool
Permanent Data Record
No changeover – less disturbance
Proficiency tool
Standardization across laboratories
Research tool
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Embryo Testing
Reprogenetics Inside
Products of Conception Analysis (POC)
Paternal Genetic Testing
Genetic Counseling
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LEADER IN PGD
COMPREHENSIVE
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GENETIC ANALYSIS
Gartner – Palmer* Hype Cycle
Progressive Use Cycle13
Current?
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GENETIC ANALYSIS
PGS 2.0
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GENETIC ANALYSIS
There is Agreement among Proponents and Opponents of PGS
on Five Findings
Many embryos are genetically abnormal
Most abnormalities occur de novo
Embryonic aneuploidy is maternal age dependent
Aneuploidy and embryonic morphology are
modestly correlated
Post-fertilization mosaicism is commonNEFS, 2015
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Why Trophectoderm Biopsy?
More cells - more DNA
Reduced learning curve
Lower error rate
Prolonged culture
Less embryos and samples to process
Single embryo transfer
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GENETIC ANALYSIS
Findings
1. Lower Error Rates
2. Higher Call Rates
3. More information (Big Data)
4. Fixation Is Less Variable
5. Less Invasive
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• Higher implantation and ongoing pregnancy
• PGS 2.0 improves embryo selection in eSET
• Significant reduced miscarriage
• Only in good prognosis patients
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Conclusions PGS 2.0
Trophectoderm biopsy appears safer
Second generation, some success & evidence
Out of Gartner’s trough - towards illumination?
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GENETIC ANALYSIS
How much further do we need to improve?
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GENETIC ANALYSIS
0
5
10
15
20
25
30
35
40
2003 2004 2005 2006 2007 2008 2009 2010
Implantation <35 years SART data from www.sart.org
Algorithms www.ivfreports.org
>400 clinics
>750,000 cycles
>2,000,000 embryos
USA National implantation rates
<35 years of age
Regression slope per year 1.46%
100% implantation reached in 38 years
AD 2053.3
Cohen et al, 2012
2011 35.9%
2012 37.4%
2013 39.4%
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GENETIC ANALYSIS
SART
implantation
success rate
<35 35-37 38-40 41-42
Improvement
per year (%)
1.46 0.93 0.64 0.32
Years to reach
100%
38 73 125 280
Year of 100%
implantation
2053 2087 2139 2294
The Future of IVF (PGS) Success Rate
(Based on SART data, Moore’s Law and without ‘paradigm shift’ – Cohen et al, 2012)
Mitochondrial DNA
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Selection of the most viable euploid blastocyst by
mitochondrial DNA quantification (Mitograde)
Euploid implanting(40%)
Aneuploid(35%)
Euploid not implanting - Unknown reason (25%)
Example: patient 35 year old, blastocyst transfer
Embryo selection – Using PGS to find viable embryos
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• Approximately 35% of euploid embryos fail to implant. Why?
• mitochondrial DNA content of embryos may be another indication of failure?
Aneuploidy is not the only reason for implantation failure
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GENETIC ANALYSISApproximately 35% of euploid embryos fail to implant. Why?
Analysis of the mitochondrial DNA content of embryos
Fragouli et al., 2015 PLoS Genetics; ASRM 2015
Explains 1/4-1/3 of implantation failures involving euploid embryos
MitoGrade
Chromosomally normal blastocysts with elevated mtDNA levels do not implant
• Mitochondria morphology and mtDNA copy number is tissue specific
• Mitochondria are maternal in origin, and contain one or more copies of their genome
• At cleavage stage the mitochondria are still those inherited from the egg
• ‘Egg’ mitochondria replicate during blastocyst formation
Blastocyst Mitochondria
• Population differences between egg and blastocyst mitochondria.
Mitochondria in preimplantation development
• M-II oocytes have high content of mitochondria and mtDNA copies (50,000-550,000)
• Ratio mtDNA/mitochondria = 1
May-Panloup et al., 2005 ; St John et al., 2010; Eichenlaub-Ritter et al., 2011; Fragouli et al., 2015. PLOS
• Mitochondria content dependent on cell’s volume & energy requirements
• Elevated mtDNA levels appear after first embryonic differentiation
• Fragouli and Wells discovered that elevated mtDNA in the blastocyst is associated with failure to implant
0
50
100
Mitochondrial genome (kb)
Se
qu
en
ce r
ead
s (d
ep
th
of
cove
rag
e)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Mitochondria quantification
Fragouli et al. (2015) PLOS
• Quantify mtDNA by qPCR or NGS
• Blastocyst biopsy
• WGA
• Targeting multiple mitochondria sites
• Normalize cell number by comparing to a multi-copy nuclear sequence
• aCGH or NGS
28% of euploid blastocysts have elevated mtDNA
Normal mtDNA levels
Elevated mtDNA levels
Pregnant Not-pregnant
Retrospective analysis
mtDNA quantity
100 euploid blastocysts
Known outcome
Fragouli et al., 2015 PLoS Genetics
0
0.005
0.01
0.015
0.02
PGS + Mitograde™: Implantation Rates
SET of euploid embryos: 68
MitoGrade™ elevated: 15.4%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
MitoGradeelevated
MitoGradeNormal
implantation
20%
86%
Reprogenetics clinical data, unpublished
NormalBlastocystmtDNA level
Egg mitochondria.
Embryonic mitochondria (replicated after genome activation)
Elevated Blastocyst mtDNA level
High implantation
Low implantation
Fragouli et al. (2015) PLOS
Approximately 35% of euploid embryos fail to implant. Why?
Analyzed the mitochondrial DNA content of embryos
Fragouli et al., 2015 PLoS Genetics; ASRM 2015
Explains 1/4-1/3 of implantation failures involving euploid embryos
MitoGrade
Chromosomally normal blastocysts with elevated mtDNA levels do not implant
Pregnant Not-pregnant0
0.005
0.01
0.015
0.02
Data obtained using the MitoGrade test
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
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 with age
Elevated mtDNA quantity 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 is associated with embryo ploidy
mtDNA quantity relationship with embryo ploidy is independent of female age
mtDNA quantity and aneuploidy
mtDNA quantity age and aneuploidy
Female age
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Embryo Selection: Conclusions
For technology lovers only
From a patient perspective: selection is desired
Improved embryology has changed the game
Ongoing improvement of implantation may be country specific
PGS 2.0 – promising, but waiting for more trials
Morphology 2.0 – QC tool, waiting for trials
mtDNA – interesting - waiting for trials
ScientistsSantiago Munné, PhD (US)Dagan Wells, PhD (UK)M. Konstantinidis, PhD (US)Mireia Sandalinas, PhD (Spain)Renata Prates (US)Samer Alfarawati, PhD (UK)Souraya Jaroudi, PhD (UAE)Tomas Escudero (US)J. Horcajadas, PhD (Latin Am.)Luis Guzman, PhD (Peru)N’Neka Goodall (US) Sophia Tormasi (US) Allen Kung (US)Lia Ribustello (US)Lauren Lansdowne (UK)Sarah Taylor (UK)Krista Miramontes (UK)Jacques Cohen, PhD (US)
Lab & Medical Directors Pere Colls, PhD (US)Carles Gimenez, PhD (Spain)Elpida Fragouli, PhD (UK)Karsten Held, MD (Germany)Tetsuo Otani, MD (Japan)Muriel Roche, PhD (Japan)Braulio Peramo, MD (UAE)Ahmed Yesilyurt, MD (Turkey)Xuezhong Zeng, MD (China)Francisco Rocha, PhD (Mexico)Christian Alvarez Sedo, PhD (Argentina)
EmbryologistsKelly KettersonCatherine WelchTim Schimmel
Genetic CouncilorsAmy JordanErin Mills
Reprogenetics Laboratories