David L. Keefe, M.D. SARG International Summit May 28,...
Transcript of David L. Keefe, M.D. SARG International Summit May 28,...
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David L. Keefe, M.D. SARG International
SummitMay 28, 2018
What Causes Genomic Instability During Preimplanation Embryo Development?
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Disclosures
• Clinical Experts Panel‐Illumina • Scientific Advisory Boards‐ Origio and Cooper Genomics
• Research funded by March of Dimes to study effects of telomere attrition on aneuploidy in human embryos
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Objectives
• Describe the pervasive genomic instability that characterizes preimplantation embryo development
• Understand how telomeres reset during preimplantation development
• Propose an explanation for genomic instability in embryos as a byproduct of telomere rejuvenation during the earliest stages of life
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I. Global Genomic Instability, Including Aneuploidy, Mosaicism & Copy Number Variants, is Common In Preimplantation Embryos
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35%
27%
18%
9%
2%
52% 50% 50.50%47.70%
50%
<35 35‐37 38‐40 41‐42 >42
SART
Euploid
Aneuploidy Contributes to Age‐Related Infertility
Harton, Grifo, Munne et al. (2013) Fertil and Steril and unpublished data.
NYU Data
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• Definition‐ presence of two or more populations of cells with different genotypes in one individual, who has developed from a single fertilized egg.[1]
[1] Stern, C. and K. Sekiguti 1931. Bio. Zentr. 51, 194–199.
What About Mosaicism?
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Does Mosaicism Invalidate PGT‐A?• Retrospective study of embryos that had undergone PGS by NGS (@ Reprogenetics) from 2/15‐1/17.
• Thawed, expanded, re‐biopsied, repeated NGS using same WGA, Veriseq, MiSeq, but in our own laboratory
• Blastocysts w/ diagnosis of mosaic, aneuploid or euploid• Whole Genome Amplification (WGA) by Sureplex• Veriseq DNA library prep • MiSeq sequencing to assess chromosomal copy number• BlueFuse Multi software• Aneuploidy‐ >80% change in chromosome copy number• Mosaicism‐ examined varying thresholds from 20‐80% (per PGDIS); also looked at 30% and 40% thresholds
Sachdev et al, ASRM 2017
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PGT‐A Is Reliable and Accurate• 129 samples from 32 blastocysts from 17 patients
• 18 mosaic, 4 aneuploid and 10 euploid• Concordance for aneuploidy‐ 97% (62/64 samples)
• Concordance b/w ICM/TE for aneuploidy‐ 100%• 2 non‐concordant samples between TE biopsies, but both showed either aneuploidy for another chromosome or complex mosaicism with more than 3 chromosomes mosaic.
.Sachdev et al, ASRM 2017
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PGT‐A Is Reliable & Accurate • Mosaic chromosome not identified consistently in repeat biopsy specimens, regardless of diagnostic criteria used to call mosaicism
• Concordance for mosaicism between clinical and experimental biopsies not affected by varying diagnostic threshold for mosaicism
• Concordance for mosaicism not different between TE and ICM
• Number of samples with additional mosaic chromosomes (a chromosome in addition to the chromosome previously diagnosed to be mosaic) significantly varied between the different diagnostic thresholds (p <0.001).
• Number of cells included in the biopsy did not affect concordance for mosaicism across all diagnostic threshold levels
Sachdev et al, ASRM 2017
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“Euploid” embryo
Aneuploid Euploid
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“Aneuploid” embryo
Aneuploid Euploid
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“Mosaic” embryoAneuploid Euploid
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Summary and Conclusions‐Mosaicism & Reliability PGS by NGS
• PGS previously thought to provide a binary output‐ normal vs. abnormal
• With increased sensitivity associated with aCGHand NGS, PGS now detects more subtle abnormalities, including mosaicism
• PGS w/ NGS provides a nuanced output, similar to glucose tolerance test for diagnosis of diabetes mellitus
• More studies needed to optimize clinical utility of PGS
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• Definition: a phenomenon in which sections of the genome are repeated and the number of repeats in the genome varies between individuals in the human population.[1] Copy number variation is a type of structural variation: specifically, it is a type of duplication or deletion event that affects a considerable number of base pairs.[2]
[1] Mccarroll et al (2007) Nature Gen 39:37‐42 [2]Sharp AJ et al (2005) Am J Hum Gen 77:78‐88
Copy Number Variants
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Copy Number Variants Are Common During Preimplantation Embryo Development
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Why So Much Genomic Instability During Early Embryo Development?
• Causes of Mosaicism‐– Mitotic non‐disjunction, leading to aneuploid cell line
– Correction of prior meiotic error
• Causes of Copy Number Variants– Anaphase lag‐ is the most common way by which mosaicism arises in the preimplantationembryo.[2]
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• Anaphase lag‐ delayed movement during anaphase, where one homologous chromosome in meiosis or one chromatid in mitosis fails to connect to the spindle apparatus, or is tardily drawn to its pole and fails to be included in the reforming nucleus.
• Chromosome forms a micronucleus in the cytoplasm and is lost from the cell.[1] The lagging chromosome is not incorporated into the nucleus of one of the daughter cells, resulting in one normal daughter cell and one with monosomy.[2]
• Anaphase lag can also cause a rescue of the daughter cell if the cell was originally trisomy.[1]
[1]Gardner, R.J.M; Sutherland, Grant R. (2004). Chromosome Abnormalities and Genetic Counseling (3rd ed.). NY: Oxford Press. p[2]Jump up^ "Human Molecular Genetics". Archived from the original on June 29, 2007.
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Anaphase Lag
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II. Telomeres & Genomic Instability in Preimplantation Embryos
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Telomere‐ Structure
• Tandem repeats (TTAGGG)• 6 proteins in a complex known as Shelterin.• Fold single stranded end of chromosome into a t‐loop to avoid DNA damage response.
• Loss of telomere leads to apoptosis or senescence.
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Telomere End Fusions Produce Anaphase Lag, Mosaicism & CNVs
Maciejowski J Nat Rev (2017)18:175‐86
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Telomere Attrition in Mice PhenocopiesReproductive Aging in Women
Increased embryo fragmentation & apoptosis(Juriscova et al, 2003)
Observation Explained by Telomere Theory
Decreased Chiasmata-(Hassold et al, 1996)
Spindle abnormalities-(Battaglia et al,1997)
Low levels mtDNA deletions & ROS -(Keefe et al, 1995)
Production Line –(Henderson and Edwards, 1968)
Keefe & Liu, 2009
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What About Women? Reproductive Function in Patient w/ Telomeropathy
Unaffected Patient (29‐31) DKC (30 years old)
Anti‐Mullerian Hormone 1‐3 0.3
Stimulation Dosage 200 IU 600IU
Stimulation Duration 10‐11 18
Oocytes Retrieved 15‐20 7
% Fertilized 90% 40%
% of Euploid embryos 50% 14%
Robinson et al, unpublished
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2007
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Telomeres Are Short in Oocytes and Elongate in Embryos‐ Even Parthenotes
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Telomere Elongation‐ Even in Telomerase Null Mice
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Robinson et al, unpublished
Telomeres Lengthen in Preimplantation Human Embryos
P = .03
N.S.
P= .03P= .0002
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P = .003
P = .019N.S.
N= 16N= 37 N= 21 N= 32
Telomeres Lengthen in Human ParthenotesFollowing Activation
Robinson et al, unpublished
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DNA Double Strand Break Repair‐ Rad 50, Bloom, Werner Expressed During Cleavage Stage
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III. What Causes Alternative Lengthening of Telomeres in Preimplantation Embryos?
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Flies Elongate Telomeres Via Retrotransposition
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Retrotransposition• With other, inactive transposable elements, e.g. Alu & Sine,
retrotransposons comprise 40% of human genome• Parasites which infected our ancestral genome over billions
of years• Line 1‐ Long Interspersed Element (L1)‐ Are active, more
recent retrotransposons‐maintain reverse transcriptase & endonuclease activities. Insert randomly throughout the genome under certain circumstances, e.g. during early embryo development.
• Comprises 17% of human genome‐ up to 100 copies active• Held in check by methylation of CpG islands in 5’ promoter
region and by histone methylation • During oogenesis & early embryo development cytosines are
demethylated to reset the epigenetic clock @ the beginning of life
• Cytosine demethylation activates L1 • Activation of L1 causes fetal oocyte attrition in mice
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Cytosine Demethylation During Gametogenesis & Early Development
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Do Mammals Mobilize Retrotransposons To Elongate Telomeres During Early Development?
Telomere RNA (TERRA)Expressed (Wang)
Telomere elongation inhibited by AZT (Navarro)
ORF2 expressed in embryos? (Bourroul)
ALT in human embryos & parthenotes(Robinson)
Methylome clock advanced in individual human oocytes? (Wang, Charmani)
Retrotransposon capture (RC) seqto compare TE architecture in euploid vs. aneupoidhuman embryos; persistence of LINE1 message in abnormal blasts? (Maxwell)
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TERRA Biogenesis in Mammals
S. Feuerhahn et al. / FEBS Letters 584 (2010) 3812–3818
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TERRA Activated in Cleavage Mouse Embryos
TERRA TRF1 DAPI Merge
Early 2cell(G1)
Late 2cell(S/G2)
4cell
A
TERRA foci per cell
>50 (n=12)
20 (n=6)
7.5 (n=6)
B
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Line1 Copy Number Increases During Early Development
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Summary & Conclusions• Genomic instability is common in preimplantation embryos
• Mitotic as well as meiotic errors‐mosaicism, copy number variants
• Telomeres are essential for genomic stability• DNA recombination‐based telomere elongation, which resembles retrotransposon activation, may contribute to global genomic instability during preimplantation embryo development
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Acknowledgements Laboratory for Human Reproduction
David L. KeefeFang ‘Helen’ WangRicardo Pimentel (Univ. Fed. De Goias) Libing Wang (UCLA)Lan Wang (Tongji Med. College)Elisa Atamian (Boston Univ.)Jason Kofinas (Kofinas Ferility) Dani Antunes (NYU FC)Molly Kumar (University of Sydney)Lin Liu (Univ. Nankai)Nidhee SachdevPaula Navarro (USP‐RP, Brazil)
NYU Fertility CenterYael KramerDavid McCullohJamie Grifo &Embryology Staff
New York Stem Cell FoundationDeter Egli
FundingMarch of DimesStanley Kaplan Research
Endowment