Nathan R. Treff PhD Chief Science Officer Detection of segmental and mosaic aneuploidy in preimplantation embryos and their reproductive potential Nathan R. Treff PhD Chief Science Officer

Frequency of mosaicism is misconceived

Primarily maternal meiotic (not mosaic) Hassold and Hunt Nature 2001 2:280

Origin of errors in preimplantation human embryos 1st polar body 2nd polar body TE biopsy maternal meiotic Treff et al Fertil Steril 2008

Origin of errors in preimplantation human embryos 1st polar body 2nd polar body TE biopsy mitotic or paternal meiotic Treff et al Fertil Steril 2008

~67% of errors from maternal meiosis Treff et al Fertil Steril 2008

Why are some mosaicism estimates so high?

Mosaicism or technical error? 0% consistency in embryos with aneuploidy!

Self correction or technical error? 0% consistency between day 3 and day 5! Kearns et al., ASRM 2010, 2011, 2012, 2013 ASHG 2011, 2012, 2013, ESHRE 2011, 2012, 2013

aCGH False Positives False Positive Rate P=0.006 n=159 embryos Capalbo et al. EJHG 2014

S-phase artifacts

Mosaicism classification schemes Capalbo et al. Hum. Reprod. In Press

Mosaicism is not common Capalbo et al. Hum. Reprod. In Press

Predictive Value of CCS? Important to know if the test can be used to safely discard an embryo

SNP array predictive value Scott et al. Fertil Steril 2012 97 870-875

tNGS predictive value None with aneuploidy Sustained Implantation Werner et al ASRM 2015

Impact of Mosaicism No method of CCS can give a correct diagnosis when testing a mosaic embryo; by definition there will always be a sampling error +15 N +15 -15 N N N +15 -15 -15 +15 -15

Mixture Model Goodrich et al. JARG 2016

Comparison of methods to detect aneuploidy in a mixture Goodrich et al. JARG 2016

↑ sensitivity = ↓ specificity Goodrich et al. JARG 2016

Mosaicism or artifact? No evaluation of specificity/false positives!

Mosaicism classification schemes Capalbo et al. Hum. Reprod. In Press

Contamination can look like mosaicism VeriSeq PGS NexCCS Copy Number Chromosome

Contamination detection (genotyping) NexCCS Copy Number Pure Chromosome Allele Ratio 1 0.66 0.5 0.33 Chromosome

Contamination detection (genotyping) NexCCS Copy Number Contaminated Pure Chromosome Allele Ratio 1 0.66 0.5 0.33 Chromosome Chromosome

Triploidy can also look like mosaicism NexCCS Triploidy can also look like mosaicism Copy Number Triploid (2:1 allele ratios) Allele Ratio 1 Diploid (1:1 allele ratios) 0.66 0.5 0.33 Allele Ratio 1 0.66 0.5 0.33

Segmental Aneuploidy

SNP array for translocations

Mitotic (reciprocal) segmental errors 5_2_B - 45,XX,+2p,+3, ++8p, -21,-22 2_2_A - 46,XY,-8,+10 1 2 3 4 1 2 3 4 5_2_C - 44,XX,-3p,-21,-22 2_2_B - 47,XY,+8 1 2 3 4 1 2 3 4 5_2_E - 44,XX,++3p,-21,-22 1 2 3 4 2_2_C – 44,XY,-8,-10 1 2 3 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Chromosome # Chromosome # 5_3_A - 46,XX,-6,+8q 1 2 3 4 1 2 3 4 1_3_F- 45,XY,-15 5_3_D - 46,XX,i(8) 1 2 3 4 1 2 3 4 1_3_C - 48,XY,++15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Chromosome # Chromosome # Kort et al. Human Reproduction. 2015

Segmental mosaicism platform comparison Frequency of Detection Mixture Level

Segmental mosaicism platform comparison

Segmental Aneuploidy Study Design Prediction of segmental error Original TE Bx TE reBx 1 TE reBx 2 TE reBx 3 ICM section Randomized blinded analysis Juneau et al ASRM 2016

Uniform segmental Juneau et al ASRM 2016

Reciprocal segmental Juneau et al ASRM 2016

Observed in original biopsy only Juneau et al ASRM 2016

Confirmed in at least one rebiopsy Juneau et al ASRM 2016

Segmental Aneuploidy Predictive Value 88% predictive of remaining embryo 60% mitotic origin artifact or true mosaicism Juneau et al ASRM 2016

Segmental aneuploidy is associated with reduced success 115 20 189 12 p = 0.0118 Werner et al ASRM 2016

Conclusions Mosaicism is overestimated by inaccurate methods and weak criteria Altered log2 ratios can originate from phenomena other than mosaicism Criteria for diagnosing mosaicism from a single biopsy needs evaluation for specificity prior to clinical utilization Segmental aneuploidy originates primarily from mitotic errors and may be predictive of reproductive potential