preimplantation genetic screening

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Preimplantation Genetic Screening

Dr Tevfik YoldemirMarmara Üniversitesi Tıp Fakültesi Kadın Hastalıkları ve Doğum A.D.

Üreme Endokrinolojisi ve İnfertilite B.D.

[email protected]

consolidated approach for single (or a few) cell genetic analysis

– i) standardization of the analytical methods,

– ii) analysis of cost-effectiveness, and

– iii) application of emerging technological

advances.

Systems Biology in Reproductive Medicine, 2012, 58: 289–300

Preimplantation genetic diagnosis is currently indicated

• autosomal recessive diseases in which both parents are known genetic carriers (cystic fibrosis, Tay-Sachs disease, and sickle cell disease),

• autosomal dominant diseases in one or both parents (Huntington’s disease),

• genetic mutations causing important consequences (BRCA gene),

• X-linked diseases (hemophilia),

• certain balanced chromosomal translocations or inversions.

Journal of Equine Veterinary Science 41 (2016) 29–34 Journal of Equine Veterinary Science 41 (2016) 29–34

Biopsy methods

Taiwanese Journal of Obstetrics & Gynecology 53 (2014) 146-150

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chromosomal mosaicism

• A systemic review by van Echten-Arends et al showed that the rate of chromosomal mosaicism of cleavage embryos appears to be as high as 72%.

• Even at the blastocyst stage, Fragouli et al demonstrated that 32.4% of blastocysts are mosaic.

• Mosaic diploid- aneuploid blastocysts with >30% normal cells account for <6% of the analyzed embryos.

• Although meiotic and postzygotic errors leading to

mosaicism are common, most mosaic blastocysts contain no normal cells.

Taiwanese Journal of Obstetrics & Gynecology 53 (2014) 146-150

chromosomal mosaicism

• Data report the rate of mosaicism at the cleavagestage of development to be as high as 50 %. Whilemosaicism does persist at the trophectoderm stage, the rate of mosaicism is markedly lower,

approximating 3-5 %, than that at earlier stages of development.

J Assist Reprod Genet (2016) 33:823–832

Methods used for molecularanalysis of all 24 chromosomes:

• metaphase comparative genomic hybridization (Wells et al.,

1999);

• array comparative genomic hybridization (aCGH) (Wells et

al., 2008; Geraedts et al., 2011);

• genome wide single nucleotide polymorphism analysis

(Harper and Harton, 2010);

• PCR-based detection (Treff et al., 2012) and

• next generation sequencing (NGS), or massive parallel sequencing (MPS), using different platforms such as the MiSeq (Fiorentino et al., 2014), the HiSeq platform (Wang et al., 2014) or the IonTorrent platform (Kung et al., 2015).

Different techniques


Trophectoderm biopsy at blastocyst stage

• (i)inter-laboratory congruity and

• (ii) intra-embryo congruity of multiple embryo biopsies in a single laboratory

• Only 2/11 (18.2 %) embryos were identically assessed at two PGS laboratories;

• 4/11 (36.4 %), on repeat analysis were chromosomally normal,

• 2 mosaic normal/abnormal, and 5/11 (45.5 %) completely differed in reported aneuploidies.

• In intra-embryo analyses, 5/10 (50 %) differed

between biopsy sites. Gleicher et al. Reproductive Biology and

Endocrinology (2016) 14:54

next-generation sequencing (NGS)

• copy number analysis for all 24 chromosomes in single or small numbers of cells, such as biopsiesfrom preimplantation embryos.

• array comparative genomic hybridization (aCGH),

• single-nucleotide polymorphism microarrays (SNP) ,

• and quantitative polymerase chain reaction (Q-PCR) .

• the focus in the PGS field has now shifted from day 3 single blastomere biopsy to day 5/6 trophectoderm(TE) sampling

Zheng et al. Molecular Cytogenetics (2015) 8:38

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next-generation sequencing (NGS)

• Chromosomal copy number assessment based on NGS may offer several advantages to aCGH including reduced DNA sequencing cost, enhanced detectionof partial or segmental aneuploidies as a result of the

potential increase in chromosomal analysis resolution, the potential automation of thesequencing library preparation to minimize human errors, reduce hands-on time, and enable higher throughput and consistency


Comprehensive chromosome screening improves embryo selection

NGS vs aCGH


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Clinical indications (n = 138) PGS with array CGH screening

• 1) unexplained recurrent pregnancy loss (URPL): patients (n = 71) with two or more unexplained miscarriages;

• 2) repeated implantation failure (RIF): patients (n = 32) with implantation failure after three or more IVF cycles or with transfer of 10 or more good morphology embryos; and

• 3) previous aneuploid conceptions (PAC): patients (n = 35) with one or more previous aneuploid conceptions (e.g. Down Syndrome).

BMC Med Genomics. 2014 Jun 22;7:38.

Clinical indications

• advanced maternal age (AMA), usually defined as maternal age above 35–38 years,

• repeated implantation failure (RIF) usually defined as three or more transfers of morphologically high-quality embryos without the establishment of pregnancy,

• recurrent miscarriage (RM) in patients with normal karyotypes (usually at least three previous consecutive miscarriages)

• and severe male factor infertility (usually defined as abnormal semen parameters).

Molecular Human Reproduction, 2016; Vol.22, No.8 pp. 539–544,

Women with unexplained RPL

• The IVF/PGS strategy had a live-birth rate of 53% and a clinical miscarriage rate of 7%. Expectant management had a live-birth rate of 67% and clinical miscarriage rate of 24%.

• The IVF/PGS strategy was 100-fold more expensive, costing $45,300 per live birth compared with $418 per live birth with expectant management.

Fertil Steril 2015;103:1215–20.

Decision making

Fertil Steril 2016;105: 188–93.

PGS - aneuploidy

Fertil Steril 2016;106: 75–9.

PGS - RCTs

Reproductive BioMedicine Online (2015) 30, 281–289

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Reproductive BioMedicine Online (2015) 30, 281–289

TE biopsy and PGS using aCGH (40-43 years)


TE biopsy and PGS using aCGH(40-43 years )


IR / LBR


PGS vs Expectant management

Human Reproduction, Vol.31, No.8 pp. 1668–1674, 2016

• PGS is to be strongly recommended when RPL is associated with miscarriages during infertility treatments, chromosomopathy in a previous miscarriage, up to five previous miscarriages and a

high incidence of chromosomal abnormalities in

spermatozoa.

RBMonline Vol 18 No 5. 2009 687-693.

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PLoS ONE 10(10): e0140779. doi:10.1371/journal.pone.0140779 PLoS ONE 10(10): e0140779. doi:10.1371/journal.pone.0140779



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Fertil Steril 2016;106:597–602

The impact of contemporary PGS• the analyzed and reanalyzed Centers for Disease Control data,

were not adequate to allow meaningful conclusions regarding the contemporary practice of PGS.

• Randomized controlled studies and cohort studies have been mainly from high quality laboratories in good prognosis patients and showed no apparent adverse effect of biopsy.

• Whether similar good outcomes can be achieved in a wide range of IVF settings and in patients with lower prognosis remains to be established.

• Deferred transfer likely has specific advantages for PGS cycles and timing and regimens for endometrial preparation are certain to be further optimized and perhaps even matched to embryo manipulation and quality.

• Intention to treat studies of deferred ET with and without PGS and eventually of all transferred embryos will be required

3G technologies

• 3G high-throughput NGS technologies are expected tooffer advantages over 2G NGS, namely:

– 1) higher throughput;

– 2) longer read lengths;

– 3) higher accuracy;

– 4) less starting DNA;

– 5) faster turnaround time;

– 6) lower cost.

• However, as found in 2G technologies, data analysis is still complex, owing to large data volume. In addition, 3G technologies need to solve signal-processing challenges. Fertil Steril 2013;99:1054–61.

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preimplantation genetic screening

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