MARINOS TSIRIGOTIS MD.FRCOG DIRECTOR “AKESO” FERTILITY CENTER

MARINOS TSIRIGOTISMD.FRCOGDIRECTOR “AKESO” FERTILITY CENTER 2nd international Meeting “New Perspectives on ovulation induction” 3-4 June 2010 Istanbul Turkey Is There any Indication for PGD?

Types of Preimplantation Genetic Testing Is There any Indication for PGD? • Preimplantation Genetic Diagnosis -PGH • Preimplantation Genetic Screening Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

What is Preimplantation genetic Testing? Is There any Indication for PGD? • The procedure which involves the removal of one or more nuclei from oocytes (polar bodies) or embryos (blastomeres or trophectoderm cells) to test for mutations in gene sequence or aneuploidy before transfer. Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

What is Preimplantation Genetic Diagnosis ? Is There any Indication for PGD? PGD • When one or both genetic parents carry a gene mutation or a balanced chromosomal rearrangement and testing is performed to determine whether that specific mutation or an unbalanced chromosomal complement has been transitted to the oocyte or embryo. Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

Indications for Is There any Indication for PGD? PGD ■ Autosomal dominant disorders (50% risk) • ■Autosomal recessive disorders (25%) • ■Female carriers of x-linked disorders • (25% of female, & ½ of male embryos) • ■Previous affected child with a de-novo mutation • (ie: Tuberous Sclerosis) • ■Human leukocyte antigen (HLA) matching • (b-thalassaemia, fanconi anaemia, ALL, DBA etc.) • ■Carriers of a balanced chromosomal translocation, inversion • or other structural chromosomal rearrangement (ie: markers) • ■Cancer predisposition Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

Is There any Indication for PGD? Autosomal dominant conditions (most frequently diagnosed) • Myotonic dystrophy • Huntington's disease • Charcot Marie tooth • Adenomatous polyposis coli • Von Hippel-Lindau • Neurofibromatosis • Marfan syndrome • Breast cancer • Tuberous sclerosis • Osteogenesis imperfecta • Retinoblastoma Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

Is There any Indication for PGD? Autosomal recessive conditions • Cystic fibrosis • β-thalassemia • Spinal muscular atrophy • Sickle cell disease • 21-hydroxylase deficiency • Tay-Sachs disease • Gaucher disease • Epidermolysis bullosa • Fanconi anemia Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

Is There any Indication for PGD? x-linked diseases • Sex-linked recessive(50% of males will be affected) • Haemophilia A • Becker muscular dystrophy • Retinitis pigmentosa • Adrenoleukodystrophy • Lesch Nyhan syndrome • Spinal bulbar muscular atrophy • Sex-linked dominant(50% of males will be affected, • females may be affected) • Duchenne muscular dystrophy • Alport syndrome • Fragile X syndrome • Incontinentia pigmenti Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

Preimplantation genetic screening (PGS) Chromosomal aneuploidy screening Is There any Indication for PGD? • When genetic parents are known or presumed to be chromosomally normal and their embryos are screened for aneuploidy. Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

Indications for PGS Is There any Indication for PGD? ■ Advanced Maternal Age (AMA) ■ Recurrent early Pregnancy Loss (RPL) (particularly with chromosomally abnormal foetuses) ■ Repeated IVF (implantation) failure (RIF) ■ Severe male infertility ■ Patients with sex chromosomes aneuploidy ■ Sex selection ■ Single embryo transfer ■ On request (patients without known genetic disorders) Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

How is PGD/PGS performed? Is There any Indication for PGD? ■ Polar Body biopsy ■ Blastomere ■ Trophectoderm biopsy Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

Is There any Indication for PGD? Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

Why Polar Body biopsy? Is There any Indication for PGD? • Polar body is found outside the ooplasm or embryo, so the embryo is not disturbed • In some countries, social and ethnical reasons do not allow for embryo biopsy • Blastomere testing for aneuplodies has an important limitation due to the extremely high rate of mosaicism at the cleavage stage Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

Indications for *Polar Body biopsy? Is There any Indication for PGD? • Maternally Derived Mendelian disease (single-gene disorders) ■Mother – carriers of chromosomal abnormalities ■Aneuploidy screening due to chromosomal pathology in AMA * Only maternally derived disorders can be detected Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

Methods of Genetic Analysis Is There any Indication for PGD? ■PCR(Polymerase Chain Reaction) for specific gene mutation disorder ■FISH (Fluorescence in situ hybridization) for aneuploidy screening & carriers of chromosomal abnormalities ■CGH (Comparative Genomic Hybridization) - array CGH - SNP array Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

Limitations ofPGD Is There any Indication for PGD? ■Relatively short interval of time available for analysis ■Only one or two cells can be analysed ■In Polymerase Chain Reaction based methods misdiagnosis or non-diagnosis from: - anucleate cell - failure of amplification of targeted DNA segment - external contamination - allele drop-out - partial amplification ■Estimated risk of transferring an affected embryo mistakenly identified as normal by PGD is • 2% for recessive disorders • 7-8% on average for dominant disorders Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

Limitations of PGS Is There any Indication for PGD? ■FISH allows evaluation of fewer than half of the 23 chromosomes pairs (X,Y, 1, 13,16,17,18,21,22) ■Up to 25% of aneuploid embryos will be found normal by FISH because the abnormal chromosomes are not among those included in the analysis ■Approximately 10% of cells removed for screening yield no results ■Splitting or diffused signals due to overlapping or misorientation of chromosomes Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

Is There any Indication for PGD? ■Failure to Hydridize ■A mosaic embryo can be better identified only if two or more cells are removed and analyzed. Exclusion of pathology requires all of the cells to be tested, destroying the embryo ■Even if two cells are removed from each embryo we can still get discordant results. Practically half of the embryos will be euploid after all cells have been analyzed Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

Is There any Indication for PGD? ■Up to 50% of those embryos identified as aneuploid at the cleavage stage will “self correct” at blastocyst stage ■ Abnormal results from FISH analysis of a single blastomere on day 3 embryo do not necessarily indicate that the embryo is abnormal ■ET at blastocyst stage Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

Limitations of PGS Is There any Indication for PGD? Comperative Genomic Hybridization (CGH) or Chromosomal Microarray Analysis (CMA) ■CGH will detect only unbalanced chromosomal changes ■Structural chromosome aberrations (reciprocal, inversions, etc) can not be detected ■Array CGH overcomes many of these limitations ■However, both CGH & array CGH do not provide information as to the ploidy. Balanced DNA content ie: tetraploid clone will appear normal Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

Limitations of PGD & PGS Is There any Indication for PGD? ■Embryologist experience ■Biopsy technique ■Spreading Technique (ie: air bubble!) ■Number of oocytes – embryos (ie: AMA, OHSS) ■Embryo morphology Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

Does biopsy affect the embryo? Is There any Indication for PGD? ■Theoretically removal of 1 or 2 blastomeres should not decrease pregnancy rates ■However, there have been reports whereby pregnancy rates have been lower after embryo biopsy compared to conventional IVF ■This may be due to the reduced implantation potential of embryos undergoing biopsy ■Biopsy technique, experience & culture conditions important co-factors Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

Is There any Indication for PGD? ■Indications “per-se” PRs ■Early growth and risk of congenital malformations not different to other ART children (ESHRE task force of PGD) ■However, PGD may pose neurological risks (memory decline and increased weight in adulthood in mice) Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

Clinical applications of PGS Is There any Indication for PGD? ■ AMA ■ RPL ■ RIF ■ Severe Male Factor infertility ■ SET ■ Patients request (unknown risk factors) Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

Is There any Indication for PGD? Clinical applications of PGS Since 2004 eleven RCTs 10 x blastomere 1 x blastocyst stage Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

PGSfor AMA Is There any Indication for PGD? ■Most of the published studies on PGS refer to AMA ■Current evidence do not support the use of PGS (aneuploid screening) to increase live birth rates in women of advanced material age due to : - increased rate of mosaicism at cleavage stage - tested cell(s) not necessarily representative of embryo - inability to examine all of the chromosomes by FISH…. ■Even with array CGH limitations regarding ploidy still exist….. Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

Is There any Indication for PGD? • However, even Array CGH has its own limitations • regarding cost, experience and ploidy difficulties Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

Is There any Indication for PGD? Clinical applications of PGS • Currently there are no conclusive data on the application of PGS regarding - repeated miscarriage, - implantation failure and - severe male factor. • In view of the lack of evidence for the effectiveness of PGS and the accumulating evidence for its harmfulness it is probably unethical to perform additional RCTs using cleavage stage biopsy. ________________ However in patients with RPL and foetuses with an abnormal chromosomal pattern PGS maybe indicated Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

Is There any Indication for PGD? Clinical applications of PGS ■Suggestions for improving results - alternative biopsy timing (PB and/or trophectoderm) - application of technologies for more comprehensive testing to include all chromosomes (CGH microarray-based testing) Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

ESHRE task force on PGS Is There any Indication for PGD? ■In order to clarify if PGS is beneficial, an RCT in AMA patients has been set up using: - Polar body biopsy - Array-comparative genomic hydridization ________________ ……hoping that the collected data will clarify the effectiveness of PGS Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

PGD for inherited predisposition to cancer Is There any Indication for PGD? ● Familial adenomatous polyposis APC ● Li-Fraumeni syndrome p53 ● Multiple endocrine neoplasia type I MEN1 ● Neurofibromatosis type 1 NF1 ● Retinoblastoma RB1 ● Von Hippel-Lindau sindrome VHL ● Breast & Ovarian cancer ● Increasing number of indications!! Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

RECOMMENDATIONS:PGD Is There any Indication for PGD? ■Before PGD is performed, genetic counseling must be provided to ensure that patients fully understand the risk for 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. ■PGD can reduce the risk for conceiving a child with a genetic abnormality carried by one or both parents if that abnormality can be identified with tests performed on a single cell. ■Prenatal diagnostic testing to confirm the results of PGD is encouraged strongly because the methods used for PGD have technical limitations that include the possibility for a false negative result. Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

RECOMMENDATIONS:PGS Is There any Indication for PGD? ■Before PGS is performed, thorough education and counseling must be provided to ensure that patients fully undersand the limitations of the technique, the risk of error and the lack of evidence that PGS improves live-birth rates as currently performed on all the conditions stated earlier (AMA, RPL, RIF, SET, SMFI etc) except in specific cases. ■Decisions concerning future treatment should not be based on the results of PGS in one or more cycles. Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

Is There any Indication for PGD? CONCLUSION Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

Yes in… Is There any Indication for PGD? • Single gene disorders • HLA matched sibling embryos to treat genetic conditions of affected children • Carriers of chromosomal abnormalities • RPL when aborted foetuses are chromosomally abnormal !! _______________ At the moment all the other indications need to be put on hold before we start routinely applying such a costly and technologically advanced technique with many inherent and external limitations Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

Future of PGD/PGS Is There any Indication for PGD? Biopsy • Polar body • Blastocyst biopsy • Vitrification PGD • New technology to allow diagnosis • Whole genome amplification • SNP arrays and array CGH PGS • PCT to see if valid procedure with clinical significance • NOT cleavage stage biopsy • Try polar body or trophectoderm • NOT FISH • Try arrays (either SNP or array CGH) J.Harper Athens PGD Symposium, MAY 29-30/2010 Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

Karyomapping Is There any Indication for PGD? • A Universal method for genome wide analysis of genetic disease based on mapping crossovers between parental haplotypes • High density genome wide single nucleotide polymorphisms (SNP) genotyping of proband parents and appropriate family member(s) to establish phase. • Mendelian analysis and Karyotyping of the parental and grandparental haplotypes for each chromosome or chromosome segment to r-chromosome Handyside A Athens PGD Symposium, MAY 29-30/2010 Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

Conclusions Is There any Indication for PGD? • Karyomapping combines accurate analysis of single gene defects and chromosome aneuploidies and partial deletions with their parental origin • The prevalence of chromosomal abnormalities in patients’ embryos underlines the importance of combined analysis for PGD • validation of new technologies !!! Handyside A Athens PGD Symposium, MAY 29-30/2010 Marinos Tsirigotis, MD.FRCOG, Director “AKESO” Fertility Center

MARINOS TSIRIGOTISMD.FRCOGDIRECTOR “AKESO” FERTILITY CENTER THANK U FOR LISTENING

Video…blastomere biopsy

Video…polar body biopsy

Video…trophectoderm biopsy

MARINOS TSIRIGOTIS MD.FRCOG DIRECTOR “AKESO” FERTILITY CENTER