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Preimplantation Genetic DiagnosisSocial Sexing
Technical Aspects
94th Clinical Genetic SeminarSR Ghaffari MSc MD PhD
M Rafati MD PhD
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Conventional Techniques
PCR-based Techniques Hybridization based techniques (FISH)
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PCR Based Techniques
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Hybridization Based Technique (FISH)
Probes Definition Types
Hybridization PrinciplesFluorescent microscopy
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Hybridization based techniqueFISH
chromosome X chromosome Y chromosome 18
male nucleus
Chromosome XChromosome Y
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Misdiagnosis
Complexity of misdiagnosis estimation due to:
o Many transferred embryos do not result in a pregnancy o Some spontaneously abort o Pregnancy termination in mistakenly predicted ones without
confirmation
(ESHRE Report, X)
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Misdiagnosis after FISH testing
Among 15981 embryo transfer: 16 (0.1%)o For chromosomal rearrangements: 0.1%o For PGS: 0.08%o For X-linked disease testing: 0.5%o For social gender selection: 0.3%
(ESHRE Report, X)
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Misdiagnosis after PCR testing
Allele dropout (ADO) and contamination are inherent pitfalls of single cell PCR and each can lead to an adverse misdiagnosis
Misdiagnosis rate of 10/3727 (0.27%) after embryo transfer for single gene disorders
Misdiagnosis rate:f 3.6% in
o (ESHRE Report, X) sex determination
FISH-based analysis, is technically more robust than a simple PCR assay for sex determination
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Social sexing: genetic method
In recent data: only FISH
No misdiagnosis reported in data XII
(ESHRE Report, XII)
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Preimplantation Genetic Screening using Next Generation Sequencing
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Chromosome abnormalities
De novoAneuploidiesSex chromosome abnormalities
Inherited Balanced chromosome abnormalitiesTranslocation Inversion …
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Aneuploidies Chromosome segregation during female meiosis is particularly error prone
in humans
It worsens with advancing age
Approximately a quarter of oocytes from women in their early 30s are chromosomally abnormal
Aneuploidy rates increasing to over 75% in the oocytes of women over 40
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Aneuploidies Most of the aneuploid embryos produced from such oocytes fail to implant in the
uterus, although a minority do succeed in forming a pregnancy only to later miscarry
It is recommended to restrict the number of embryos transferred to the uterus, ideally a single embryo
Currently, the decision of which embryo should be transferred is primarily based on a simple evaluation of morphology.
However, the appearance of an embryo is only weakly correlated with its potential to form a pregnancy and reveals no useful information about its chromosomal status
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PGS In the past decade
PGS by FISH Limited number of studied chromosomes (22, 16, 21, 18, 13, X, Y, …)
Meta analysis: no improvement in pregnancy rate
In recent 2 years Array based techniques
Screening of all 24 chromosomes several randomised trials chromosome screening have been undertaken, producing
clinical data supporting the hypothesis that screening of embryos for aneuploidy can improve IVF outcomes, increasing pregnancy rates and reducing miscarriages
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Method
Three patient groups are compared: Couples with RIF for whom embryos were selected by array CGH (group RIF-
PGS): 43 Couples with the same history for whom array CGH was not performed (group RIF
NO PGS): 33 Good prognosis infertile couples with array CGH selected embryos (group NO RIF
PGS): 45
A single euploid blastocyst was transferred in groups RIF-PGS and NO RIF PGS. Array CGH was not performed in group RIF NO PGS in which 1-2 blastocysts
were transferred
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Results
One monoembryonic sac with heartbeat was found in: 28 patients of group RIF PGS (68.3%) 31 patients of group NO RIF PGS (70.5%)
In contrast, an embryonic sac with heartbeat was only detected in 7 (21.2%) patients of group RIF NO PGS.
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Conclusion
PGS by array CGH with single euploid blastocyst transfer appears to be a successful strategy for patients with multiple failed IVF attempts.
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Limitations
The extremely limited amount of tissue available for analysis, only one cell (blastomere)
Cost: cost of aneuploidy screening is multiplied by the number of embryos produced
by the couple (averaging approximately six, but in some cases exceeding 20)
Time Scalability: any test must be scalable, allowing multiple samples to be
assessed simultaneously Less than 24 h available for genetic analysis.
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Method
A rapid (<15 h) NGS protocol was developed, with consumable cost only two-thirds that of the most widely used method for embryo aneuploidy detection
Validation involved blinded analysis of 54 cells from cell lines or biopsies from human embryos.
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Results
Research Phase: Sensitivity and specificity were 100%.
Clinical phase: The method was applied clinically, assisting in the selection of euploid
embryos in two IVF cycles, producing healthy children in both cases.
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Simultaneous Investigation of Single Gene Disorders
NGS allows the potential for simultaneous chromosomal analysis and diagnosis of gene mutations in single cells Aneuploidy: low-pass sequencing of WGA products yields <0.1%
coverage of the genome PCR before NGS Library preparation Combination of libraries NGS
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Conclusion
This study demonstrates that NGS provides highly accurate, low-cost diagnosis of aneuploidy in cells from human preimplantation embryos and is rapid enough to allow testing without embryo cryopreservation.
The method described also has the potential to shed light on other aspects of embryo genetics of relevance to health and viability.
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Detection of Chromosome Abnormalities using NGS
Our Experience
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Investigation of products of conception
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Product of conception: Trisomy of chromosome 16
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Product of conception: Trisomy of chromosome 22
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Detection of unbalanced chromosome abnormality
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Partial trisomy of 1qpartial monosomy of 4p
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Preimplantation genetic screening using NGS (PGS-NGS)
Aneuploidy screening of all chromosomes Increasing the pregnancy rate following ART Techniques Proposed clinical applications:
Repeated ART Failure Recurrent abortion Routine ART ….
Workflow: Single cell whole genome amplification of blastomeres NGS analysis
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Whole Genome Amplification
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Rule out of Contamination
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STR Markers
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Fetus and the Parents
• M
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Normal embryo
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PGS-NGSMonosomy of chromosome 13
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Gender: Female
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Gender: Male
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ESHRE Report
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ESHRE PGD
European society of human reproduction and embryology Established in 1997
The aims: (i) to survey the availability of PGD (ii) to collect prospectively and retrospectively data on accuracy, reliability and
effectiveness of PGD (iii) to initiate follow-up studies (iv) to produce guidelines and recommended PGD protocols (v) To formulate a consensus on the use of PGD (vi) to educate in the science of genetics and reproduction
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Pregnancies and Babies
6458 fetal sacs 5187 clinical pregnancies 744 pregnancy complications 4140 deliveries 5135 newborns (singletons: 3182, twins: 921, triplets: 37) PND in 2462 cases and postnatal investigation in 2049 cases
(ESHRE Report, X)
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Pregnancies and Babies Till 2013:
50000 cycles10000 babies
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What about PGD babies?
Among 4021 newborns:Major malformations: 84 (2%)Neonatal complications: 402 (10%)
These cumulative data again confirm that pregnancies and babies born after PGD are similar to the pregnancies obtained and babies born after ICSI treatment
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Final Recommendation
• Before PGD is performed, genetic counseling must be provided to ensure that patients fully understand the risk of having an affected child, the impact of the disease on an affected child and the limitations of available options that may help to avoid the birth of an affected child.
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Thank You
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