The Future of Pre-implantation Genetic Diagnosis :The Future of Pre-implantation Genetic Diagnosis Dr. S. Selva MBBS (Malaya ) FRCOG (UK) FICS (USA), Master of Reproductive Medicine (Sydney) Consultant Obstetrician and Gynaecologist, Mahkota Medical Centre, Melaka, Lecturer, Melaka Manipal Medical College, Visiting Minimally Invasive Surgeon, Melaka Hospital.


What is Preimplantation Genetic Testing? :What is Preimplantation Genetic Testing? The procedure which involves the removal of one or more nuclei from oocytes (polar bodies) or embryos (blastomeres or trophoectoderm cells) to test for mutations in gene sequence or aneuploidy before transfer.


Types of Preimplantation Genetic Testing :Types of Preimplantation Genetic Testing Preimplantation Genetic Diagnosis Preimplantation Genetic Screening


What is Preimplantation Genetic Diagnosis (PGD)? :What is Preimplantation Genetic Diagnosis (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 transmitted to the oocyte or embryo.


Indications for PGD :Indications for PGD Autosomal dorminant (50% risk) Autosomal recessive (25% risk) Female carriers of X-linked disorders (25% - half of the male embryos) Carriers of mutations Human leukocyte antigen (HLA) matching Carriers of a balanced chromosomal translocation, inversion, or other structural chromosomal rearrangement


Preimplantation genetic screening (PGS) :Preimplantation genetic screening (PGS) When the genetic parents are known or presumed to be chromosomally normal and their embryos are screened for aneuploidy


Indication for PGS :Indication for PGS Women of advanced maternal age History of recurrent early pregnancy loss Repeated IVF failure Severe male infertility Sex selection


How is PGD/PGS performed? :How is PGD/PGS performed? Polar Body biopsy Blastomere Trophodecterm biopsy


Advantages of Polar Body biopsy :Advantages of Polar Body biopsy Polar body is found outside the embryo and so the embryo is not disturbed. The worry of embryo damage does not occur In some countries, social and ethical reasons does not allow for embryo biopsy Blastomere testing for aneuplodies has an important limitation due to the extremely high rate of mosacism rate at the cleavage stage.


Indications for Polar Body biopsy? :Indications for Polar Body biopsy? Maternally Derived Mendelian disease (single- gene disorders) 1) Specific diagnosis in X- linked disorders 2) PGD for couples with homozygous or double-heterozygous affected female partners 3) PGD for couples with male and female partners carrying different mutations of the causative gene 4) PGD combined with human leukocyte antigen (HLA)-typing for detecting mutations –free maternal-matched oocytes 5) PGD combined with aneuploidy testing for detecting embryos free of maternal mutation to be tested further by FISH


Indications for Polar Body biopsy? :Indications for Polar Body biopsy? For chromosomal disorders 53% of oocytes from patients of advanced reproductive age undergoing IVF are aneuploid


Methods of Genetic Analysis :Methods of Genetic Analysis For specific gene mutations PCR – Polymerase Chain Reaction- to amplify a segment of genome that contains the specific gene of interest FISH – Fluorescence in situ hybridization –DNA probes labeled with distinctly coloured fluorochromes that bind to specific DNA sequences unique to each chromosome. FISH is used to detect missing or excess chromosomal material in oocytes (when a female partner is the carrier) or embryos (when either partner is the carrier) or individuals known to habour a balanced chromosomal translocation or other structural rearrangement.


What are the technical problems of performing PGD? :What are the technical problems of performing PGD? Relatively short interval of time available for analysis Only one or two cells can be analysed


Technical problems :Technical problems In Polymerase Chain Reaction (PCR) based methods misdiagnoses from anucleate cell failure of amplification of targeted DNA segment external contamination allele drop-out – when one allele for the gene of interest fails to amplify partial amplification


Technical Problems :Technical Problems Estimated risk of transferring an affected embryo mistakenly identified as normal by PGD is 2% for recessive disorders 11% for dorminant disorders


Does biopsy affect the embryo? :Does biopsy affect the embryo? Theoretically removal of 1or 2 blastomeres should not decrease pregnancy chances However world wide, Pregnancy rates has been lower after embryo biopsy compared to conventional IVF This may be due to the reduced implantation potential of embryos undergoing biopsy.


Rationale for Preimplantation Genetic Screening? :Rationale for Preimplantation Genetic Screening? Aneuploidy is the most common cause for early pregnancy failure. The prevalence of oocyte and embryo aneuploidy increases with a) maternal age b) chromosomally normal couples with recurrent early pregnancy loss c) repeated IVF failure despite transferring of high quality embryos (based on morphology)


Technical Difficulties :Technical Difficulties FISH allows evaluation of fewer than half of the 23 chromosomes pairs in a single cell (X, Y 1, 13,16,17,18,21) Studies have shown that up to 25% of aneuploid embryos are judged normal by FISH because the abnormal chromosome pair(s) were not among those included in the analysis. Comparative genomic hybridization (CGH) can be used to test all 23 chromosomes but takes longer time and so requires embryo cryopreservation


Technical Difficulties :Technical Difficulties Approximately 10% of cells removed for screening yield no results Erroneous results maybe obtained when the orientation or overlapping of chromosomes yields split or diffused signals that are misinterpreted


Technical Difficulties :Technical Difficulties Aneuploidy may arise in several ways When non-disjunction occurs during meiosis, all of the cells in the embryo typically are aneuploid. In contrast, mitotic non-disjunction yields two or more distinct cell lines and results in an embryo that contains both normal and abnormal cells; the actual proportions of normal and abnormal cells will vary, depending on the point at which the abnormal segregation occurred. A mosaic embryo can be identified only if two or more cells are removed and analyzed. Even then, the true proportions of normal and abnormal cells cannot be determined unless all of the cells are analyzed, thus destroying the embryo.


Technical Difficulties :Technical Difficulties One study, in which the two cells removed from each embryo yielded discordant results (one normal and one abnormal), found that half of the embryos were euploid after all cells were analyzed Evidence suggests that up to half of all embryos identified as aneuploid at the cleavage stage and that survive to the blastocyst stage will ‘‘self correct’’. In summary, an abnormal result from FISH analysis of a single blastomere removed from a day 3 embryo does not necessarily indicate that the embryo is abnormal and ill-fated.


Does PGS improves pregnancy rates in advanced maternal age? :Does PGS improves pregnancy rates in advanced maternal age? Gianaroli L, Magli MC, Ferraretti AP. Preimplantation diagnosis for aneuploidies in patients undergoing in vitro fertilization with a poor prognosis: identification of the categories for which it should be proposed. Fertil Steril 1999;72:837–44. Staessen C, Platteau P, Van Assche E, Michiels A, Tournaye H, Camus M, et al. Comparison of blastocyst transfer with or without preimplantation genetic diagnosis for aneuploidy screening in couples with advanced maternal age: a prospective randomized controlled trial. Hum Reprod 2004;19:2849–58. Mastenbroek S, Twisk M, Echten-Arends J, Sikkema-Raddatz B, Korevaar JC, Verhoeve HR, et al. In vitro fertilization with preimplantation genetic screening. N Engl J Med 2007;357:9–17.


Does PGS improves pregnancy rates in advanced maternal age? :Does PGS improves pregnancy rates in advanced maternal age? Current evidence does not support the use of PGS (aneuploidy screening) to increase live birth rates in women of advanced maternal age.


Does PGS improves pregnancy rates in Recurrent pregnancy loss? :Does PGS improves pregnancy rates in Recurrent pregnancy loss? Current evidence does not support the use of PGS for patients with RPL because it does not improve ongoing pregnancy or live-birth rates and does not decrease miscarriage rates in such women. However, couples in whom RPL can be attributed to a balanced translocation may indeed benefit from specific genetic testing (PGD) to detect excess or missing genetic material in their embryos. Rubio C, Simon C, Vidal F, Rodrigo L, Pehlivan T, Remohi J, et al. Chromosomal abnormalities and embryo development in recurrent miscarriage couples. Hum Reprod 2003;18:182–8. Platteau P, Staessen C, Michiels A, Van Steirteghem A, Liebaers I, Devroey P. Preimplantation genetic diagnosis for aneuploidy screening in patients with unexplained recurrent miscarriages. Fertil Steril 2005;83:393–7. Ferraretti AP, Magli MC, Kopcow L, Gianaroli L. Prognostic role of preimplantation genetic diagnosis for aneuploidy in assisted reproductive technology outcome. Hum Reprod 2004;19:694–9.


Does PGS improves pregnancy rates in Repeated implantation failures? :Does PGS improves pregnancy rates in Repeated implantation failures? Present available evidence does not support the use of PGS for patients with repeated implantation failure. Pehlivan T, Rubio C, Rodrigo L, Romero J, Remohi J, Simon C, Pellicer A. Impact of preimplantation genetic diagnosis on IVF outcome in implantation failure patients. Reprod Biomed Online 2003;6:232–7. Munne S, Sandalinas M, Escudero T, Velilla E, Walmsley R, Sadowy S, et al. Improved implantation after preimplantation genetic diagnosis of aneuploidy. Reprod Biomed Online 2003;7:91–7. Gianaroli L, Magli MC, Ferraretti AP. Preimplantation diagnosis for aneuploidies in patients undergoing in vitro fertilization with a poor prognosis: identification of the categories for which it should be proposed.Fertil Steril 1999;72:837–44.


Does PGS improves pregnancy rates in Male Factor infertility? :Does PGS improves pregnancy rates in Male Factor infertility? Available evidence does not support the use of PGS for couples receiving IVF/ICSI for male factor indications at this time. Male Infertility Best Practice Policy Committee of the American Urological Association; Practice Committee of the American Society for Reproductive Medicine. Report on optimal evaluation of the infertile male. Fertil Steril 2004;82(Suppl 1):S123–30. Calogero AE, De Palma A, Grazioso C, Barone N, Romeo R, Rappazzo G, et al. Aneuploidy rate in spermatozoa of selected men with abnormal semen parameters. Hum Reprod 2001;16:1172–9.


PGS for Gender Selection? :PGS for Gender Selection? Patients undergo embryo biopsy with a different goal in mind and do not necessarily attempt to maximize IVF pregnancy chances With gender selection being the primary goal, conception chances come second


Conclusions :Conclusions


RECOMMENDATIONS: PGD :RECOMMENDATIONS: 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.


RECOMMENDATIONS: PGD :RECOMMENDATIONS: PGD 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.


RECOMMENDATIONS: PGD :RECOMMENDATIONS: PGD 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


RECOMMENDATIONS: PGS :RECOMMENDATIONS: PGS Before PGS is performed, thorough education and counseling must be provided to ensure that patients fully understand the limitations of the technique, the risk of error, and the lack of evidence that PGS improves live-birth rates


RECOMMENDATIONS: PGS :RECOMMENDATIONS: PGS Available evidence does not support the use of PGS as currently performed to improve live-birth rates in patients with advanced maternal age.


RECOMMENDATIONS: PGS :RECOMMENDATIONS: PGS Available evidence does not support the use of PGS as currently performed to improve live-birth rates in patients with previous implantation failure.


RECOMMENDATIONS: PGS :RECOMMENDATIONS: PGS Because the prevalence of aneuploidy is high in the embryos of patients with recurrent implantation failure, decisions concerning future treatment should not be based on the results of PGS in one or more cycles


RECOMMENDATIONS: PGS :RECOMMENDATIONS: PGS Available evidence does not support the use of PGS as currently performed to improve live-birth rates in patients with recurrent pregnancy loss


RECOMMENDATIONS: PGS :RECOMMENDATIONS: PGS Available evidence does not support the use of PGS as currently performed to reduce miscarriage rates in patients with recurrent pregnancy loss related to aneuploidy.


Slide 42:Thank You