Mitochondrial DNA levels and aneuploidy, Jacques Cohen, Reprogenetics

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Mitochondrial DNA levels and aneuploidy

a new system to assess implantation potential

Jacques Cohen(Reprogenetics)

SWES, 2016


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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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Points to cover

Is embryo selection desired?

Hype and hype cycle

Quantifying mtDNA (mitograde)


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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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Is Embryo Selection Desired?


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Technology Lovers - Banksy


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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!


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Introducing new technology, such as time-lapse, PGS 2.0 and qt mtDNA

(mitograde)


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Gartner – Palmer* Hype Cycle

11


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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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Gartner – Palmer* Hype Cycle

Progressive Use Cycle13

Current?


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PGS 2.0


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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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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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How much further do we need to improve?


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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%

http://www.sart.org

http://www.ivfreports.org


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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

[email protected]

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

Mitochondrial DNA levels and aneuploidy, Jacques Cohen, Reprogenetics

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