Mosaicism in PGT-A Is it a red herring?

Nathan R. Treff PhD, HCLD(ABB)

Cofounder, Chief Science Officer, and Clinical Laboratory Director

Disclosures

• Cofounder, Shareholder and CSO, Genomic Prediction Inc

• Director, Genomic Prediction Clinical Laboratory Inc

Stephen Hsu Laurent Tellier

SNP array (18)

qPCR (22) NGS

(19)

PGS 2.0 Mosaicism Publications “Have you published anything?”

“You’re cherry picking data”

“Do you have your own clinic?”

“You’re being dishonest”

“You can’t criticize a NEJM paper”

“You didn’t read the supplementary data”

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Treff Wells Fiorentino Munne

Debating Mosaicism

Truth

Only conflict of interest?

Laboratory Regulations Conflict of Interest

X X i.e. Germany

Laboratory Quality Conflict of Interest

Suboptimal Culture

Commercial Competition Conflict of Interest

Key Points

• Mosaicism exists, but is rare

• General lack of acknowledgement of technical limitations and artifacts

• Clinical diagnosis from a single biopsy provides poor predictive value

• Validation is extremely limited

Post implantation-primarily maternal meiotic (not mosaic)

Hassold and Hunt Nature 2001 2:280

n=358

Preimplantation-primarily maternal meiotic (not mosaic)

Why are some mosaicism estimates so high?

No meiotic aneuploidy?

Mosaicism or technical error?

aCGH False Positives

n=159 embryos

P=0.006

False Positive

Rate Single Cell < Multi Cell

Mosaicism or technical error?

9% FPR= 47% “mosaicism”

Mosaicism classification schemes

3-20%

Mosaicism is not common

~6%

Positive Controls?! (Mixture Model)

Comparison of methods to detect aneuploidy in a mixture

↑ sensitivity = ↓ specificity

33% False Positive Rate

Alternative Explanations

False Positive Mosaicism?

False Negative Uniform

Aneuploidy?

What is the true positive rate/predictive value?

?

?

?

Preimplantation stage

rebiopsy Amnio/CVS?

0 true positives Postimplantation stage

Mosaic

Predictive value for remaining embryo

~one third of embryos with a mosaic range chromosome were actually uniform aneuploid

<3% mosaicism rate on >20,000 embryos

Predictive value for remaining embryo

False negative uniform aneuploidy

Mosaic range trisomy 13

Uniform trisomy 13

Uniform trisomy 13

False negative uniform aneuploidy

Disomy

Trisomy

Mosaic +6, -13, +16?

Near Triploid Not Mosaic (using Allele Ratios)

AAA

AAB

ABB

BBB

AB

BB

AA

Allele Ratio

Mosaic +6, -13, +16 70,XXX,+6,-13,+16

AAAA

AAAB

ABBB

BBBB

AABB

AAAA

AAAB

ABBB

BBBB

AABB

Mosaicism Reporting

88% of clinics opt out of reporting mosaicism

74% of clinics do not use mosaicism testing

Non-invasive mosaicism prediction?

Micronuclei and genuine mosaicism

Time-lapse characterization of micronuclei?

Conclusions • Mosaicism exists, but is overestimated by imperfect methods and lax criteria

• Intermediate copy number can originate from phenomena other than mosaicism

• Results from a single biopsy should not be considered a definitive diagnosis of mosaicism

• Make raw data available

• Publish studies with rebiopsies of embryos classified as mosaic

• Perform systematic analysis of products of conception after transfer of “mosaic” embryos

• Develop time-lapse integrated analysis (machine learning algorithms)

Future Directions

References Cited

• Rosenwaks et al Fertil Steril 2018

• Hassold and Hunt Nature 2001

• Treff et al Fertil Steril 2008

• Mertzanidou et al Hum Reprod 2013

• Capalbo et al EJHG 2015

• Capalbo et al Hum Reprod 2017

• Goodrich et al JARG 2017

• Greco et al. NEJM 2015

• Scott et al Fertil Steril 2012

• Sachdev et al Fertil Steril 2017

• Katz-Jaffe et al Fertil Steril 2017

• Marin et al Curr Op OBGYN 2017

• Joshi et al Fertil Steril 2018

• Kim et al Am J Obstet Gynecology 2018

• Hong et al Fertil Steril 2014

• Kort et al Hum Reprod 2016

• Chavez et al Nature Comm 2012

Bold = Treff coauthor