Comprehensive Chromosome Screening Is NextGen Likely to be the Final Best Platform and What are its Advantages and Quirks?

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Horatio Gallagher
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1 Comprehensive Chromosome Screening Is NextGen Likely to be the Final Best Platform and What are its Advantages and Quirks? Embryo 1 Embryo 2 combine samples for a single sequencing chip Barcode 1 CTAAGGTAAC Barcode 2 TAAGGAGAAC Richard T. Scott, Jr, MD, HCLD Clinical and Scientific Director, Reproductive Medicine Associates of New Jersey Professor and Fellowship Director, Reproductive Endocrinology Sidney Kimmel Medical College, Thomas Jefferson University Scott et al Fertility and Sterility 2013; 100:

100 90 80 70 % 60 50 40 With greater experience, actual negative predictive value is ~98.

embryos Clinical Error Rate Per embryo 0.2% Per transfer 0.3% Per ongoing pregnancy 0.1% 3168 transfers 2976 gestations (62.

2 % With greater experience, actual negative predictive value is ~98.8% < Age (yrs) Scott et al Fertil Steril 2012; 97:870 5 Clinical Experience Misdiagnoses 4974 embryos Clinical Error Rate Per embryo 0.2% Per transfer 0.3% Per ongoing pregnancy 0.1% 3168 transfers 2976 gestations (62.1%) 10 errors 1 tetraploid 2 monosomies 7 trisomies 2354 ongoing / delivered (72.1%) Mean age 38.4 years 10 errors 7 found in losses 3 found in ongoing preg. Mosaicism evaluated in 4 samples 100% mosaic

3 How NextGen Sequencing Works Amplification of limited starting material Millions of small fragments are generated Embryo Biopsy NextGen Sequencing Alignment Aligned relative to a reference genome

4 Next Generation Sequencing Depth of Coverage Assemble all the fragments The number of fragments which fit into a given area of the genome defines the DEPTH High number of fragments allows each base pair to be sequenced/ evaluated thousands of times in a single assay depth Next Generation Sequencing Copy Number Analyses Monosomy Trisomy

5 Next Generation Sequencing Reality There are over three billion base pairs in the human genome There are many fewer reads per chip. Then add multiple embryos per chip. What percentage of the genome is covered with typical coverage following Whole Genome Amplification (WGA) and Next Generation Sequencing?.. <5% and typically much <1% Next Generation Sequencing Dealing with the inevitable lack of coverage WGA Divide each chromosome into a number of bins Assign each segment to a bin Count the number of mapped pieces which fall into each segment Compare and assign a copy number to that bin Integrate to assign a copy number for the chromosome ~150,000 integrated data points Numerically similar to small SNP arrays What size box is optimal? 50k 20k 10k

NextGen and SNP Array A comparison of cell lines with known abnormalities Yin et al Biol Reprod 2013; 88:(69), 1 6 Next Generation Sequencing Dealing with the inevitable lack of coverage Targeted

6 NextGen and SNP Array A comparison of cell lines with known abnormalities Yin et al Biol Reprod 2013; 88:(69), 1 6 Next Generation Sequencing Dealing with the inevitable lack of coverage Targeted Amplification Uses Taq based chemistry which amplifies uniformly Sequencing continues beyond the starting sequence and the remainder is locus specific which allows mapping Map each target to the appropriate locations on each chromosome Compare and assign a copy number to each location Integrate to assign a copy number for the chromosome

7 The Economics of NextGen and A Major Factor for Accuracy NextGen Sequencing Chip $$$$$$$ NextGen Molecular Barcoding Reduced Costs Embryo 1 Embryo 2 combine samples for a single sequencing chip Barcode 1 Barcode 2 CTAAGGTAAC TAAGGAGAAC

8 The Economics of NextGen A Major Factor for Accuracy NextGen Sequencing Chip $$$$$$$/2 The Economics of NextGen A Major Factor for Accuracy NextGen Sequencing Chip $$$$$$$/4

9 The Economics of NextGen A Major Factor for Accuracy NextGen Sequencing Chip $$$$$$$/48 The Economics of NextGen A Major Factor for Accuracy NextGen Sequencing Chip 96 or more $$$$$$$/96

10 Whole Genome Sequencing based NextGen (16 per chip) known trisomy copy number known monosomy unpublished data chromosome Whole Genome Sequencing based NextGen (48 per chip) copy number unpublished data chromosome

11 Targeted Amplification based NextGen (96 per chip) copy number unpublished data chromosome Chromosome specific cutoffs 4 NGS based copy number on chr chr16 specific cutoffs 1 0 unpublished data

NextGen CCS A non selection study Sustained Implantation Rate (%) 70 60 50 40 30 20 10 0 97/146 Euploid

001 0/41 187 embryo transferred Trophectoderm biopsy Transfer prior to any analysis of the biopsy Follow up to

12 NextGen CCS A non selection study Sustained Implantation Rate (%) /146 Euploid Aneuploid P< / embryo transferred Trophectoderm biopsy Transfer prior to any analysis of the biopsy Follow up to determine the predictive value of the aneuploidy result Nextgen based CCS Targeted amplification Ion Torrent sequencing Werner et al ASRM 2015 High concordance High result rate Used the same post amplification product

13 Micro Duplication Grifo et al ASRM 2015 Different Diagnoses with acgh and NextGen Grifo et al ASRM 2015

Aneuploid Blastocysts November 19, 2015 Greco E, Minasi MG,

14 Overall Euploidy Rates acgh versus NextGen Grifo et al ASRM 2015 Healthy Babies after Intrauterine Transfer of Mosaic Aneuploid Blastocysts November 19, 2015 Greco E, Minasi MG, Fiorentino F Correspondence to the editor Journal Club April 19, 2016

15 Array CGH Based Detection of Embryonic Mosaicism Array CGH Based Detection of Embryonic Mosaicism

001 Fragouli et al ASRM 2015 Healthy Babies after Intrauterine Transfer of Mosaic

16 Outcomes of Euploid and Mosaic Embryos NGS N Miscarry Ongoing Mosaic 43 5 (12%) 11 (26%) Euploid 51 5 (10%) 26 (51%) P<0.001 Fragouli et al ASRM 2015 Healthy Babies after Intrauterine Transfer of Mosaic Aneuploid Blastocysts 18 embryo transfers 6 deliveries 2 early losses 10 not pregnant Greco E et al. N Engl J Med 2015;373:

17 Single Cell Reconstitution Data +3 SD Trisomy Disomy Disomy Ovelap with Mosaic Range is ~ the range of the 2 3 SD s from mean Greco et al NEJM 2015 Single Cell Reconstitution Data +3 SD Trisomy Disomy Greco et al NEJM 2015

18 Back to Basics The impact of prevalence on Predictive Values Approximately 2.1% of euploid embryos will have results between 2 and 3 SD above the mean Normal Distribution The impact of random variation relative to standard deviations of the distribution Dramatic impact on the Predictive Values of a test when the prevalence of an abnormality if relatively low. Understanding a Mosaic Range Result in CCS Greco normal curve demonstrates that approximately 35% of mosaics will fall in the disomic range Results which are above trisomic threshold are typically designated trisomy even though some are likely mosaic Start with 1000 euploid embryos.. High Mosaic Range and above Disomic Mosaic 1 17 Mosaic Range Disomic Range Total Mosaic Range Expectations Disomic 19/32 59% Mosaic 13/32 41% Expectation: ~60% of normal implantation rates

19 What does a Mosaic Range Result Mean? True State of Nature Mosaic Disomic Predictive Value of Mosaic Result for Mosaicism: 27 / (27+43) = 38.5% Lab Result Mosaic Result Non Mosaic Range What are the anticipated delivery rates following eset? Assume no mosaic implantations 60% of the 43 = 26 Overall implantation rate of 26/ 70 = 37% Duplications and Deletions why worry? Arrays versus metaphase spread at time of amniocentesis Wopner et al NEJM December 2012 Population mostly advanced maternal age Duplications and Deletions Evaluated 80 dup/del s with known clinical phenotype 1 in 72 fetus s had a clinically significant dup/del! Risk considerably higher than for ongoing aneuploid gestation Possible to simultaneously screen for these dup/del s and aneuploidy with multiple platforms Extensive validation required

Detection of Known Duplications or Deletions in Established Cell Lines with Veriseq % Detection 100 90 80 70 60 50 40 30 20 10 0 1.4 2.5 5 6 16.3 25.

re biopsied four times to determine if the original dup/del could be confirmed Confirmed in all biopsies meiotic Confirmed in some but not all biopsies mitotic

20 Detection of Known Duplications or Deletions in Established Cell Lines with Veriseq % Detection Deletion or Duplication Size (MB) Treff et al ASRM 2015 Re evaluation of embryos with evidence of a dup/del on initial TE biopsy 28% 12% 32% 28% 32 embryos re biopsied four times to determine if the original dup/del could be confirmed Confirmed in all biopsies meiotic Confirmed in some but not all biopsies mitotic Reciprocal error in some biopsies mitotic Not found in other biopsies Scott KL et al in review 88% of initial dx confirmed Other 12% low level mosaics vs analytical errors

The Clinical Significance of Dup / Dels Next Gen using a Universal Primer and Ion Torrent Sequencing Sustained Implantation Rate (%) 70 60 50 40 30 20 10 0 P<0.

21 The Clinical Significance of Dup / Dels Next Gen using a Universal Primer and Ion Torrent Sequencing Sustained Implantation Rate (%) P< Dup / Del Euploid Werner et al in review Lower limit of detection ~ 5 MB Mitochondrial Density Additional Parameter to Aid Selection? WGA amplification Mitochondrial Density Normalized against Alu s to control for number of cells TE biopsy Higher density associated with lower outcomes Delivered Failed

22 mtdna copy number is not predictive of reproductive potential among sibling embryos Paired design Evaluates test in setting where it will be applied eset inappropriate for these types of questions Delivered Not Delivered Not useful as an adjunct to embryo selection Does Embryo Biopsy Impact the Developmental Potential of the Oocyte Routine IVF Care through Retrieval Transfer the embryos Identify mature oocytes ICSI, culture, and select 2 best embryos for transfer Implantation, Maternal serum sampling for free fetal DNA and Fingerprinting Cell submitted for eventual aneuploidy screening and fingerprinting One embryo randomized to undergo biopsy N=113 pairs; 226 embryos

23 Overall implantation rates 39% reduction insignificant 27% (mean maternal age 32) reported by Gutierrez Mateo, C., et al. Fertility and sterility 92, (2009) Evaluating the TE Biopsy Size Neal et al in review

Evaluating the TE Biopsy Size 100 90 N=1147 Percent 80 70 60 50 40 30 20 10 * P<0.

24 Evaluating the TE Biopsy Size N=1147 Percent * P<0.01 Q1 Q2 Q3 Q4 0 Chemical pregnancy Implantation Ongoing pregnancy Live birth Outcome Neal et al in review Impact of CCS and Synchrony on Clinical Implantation Rates

New methods for embryo selection: NGS and MitoGrade

New methods for embryo selection: NGS and MitoGrade Santiago Munné, PhD US: Livingston, Los Angeles, Chicago, Portland, Miami / Europe: Barcelona (Spain), Oxford (UK), Hamburg (Germany) / Asia: Kobe (Japan),