Overview

Organization of the PGD Centre

Overview of Preimplantation Genetic Diagnosis

The potential transmission of genetic disorders in couples contemplating pregnancy has always been a major problem.In the last decades, careful evaluation of family history, karyotyping, , age of the mother and more recently the age of the father and genetic screening for some of the most common genetic diseases have greatly decreased the incidence of affected pregnancies.

What is preimplantation genetic diagnosis (PGD)?

The embryo is created via in vitro fertilization.

Typically, a single cell is removed from the embryo at the 8-cell stage (3 days after fertilization).

Genetic testing is performed.

The results of testing are used to decide which embryos, if any, to implant in the prospective mothers uterus.

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PGD:Pre-implantation Genetic Diagnosis

Schwartz 2011 Jewish News

What % of IVF clinics provide testing for the following reasons?

aneuploidyautosomal disorderschromosomal rearrangementX-linked diseasesnon-medical sex selectionavoid adult-onset diseaseHLA typingHLA typing w/o single gene testSelect for a disabilityhttp://www.dnapolicy.org/resources/GeneticTestingofEmbryos.pdf

5Aneuploidy an atypical number of chromosomes, which causes disorders such as Downs Syndrome.

Baruch, S., D. Kaufman, and K. Hudson. 2008. Genetic Testing of Embryos: Practices and Perspectives of U.S. IVF Clinics. Fertility and Sterility 89: 1053-1058.

Public attitudes regarding acceptable uses of PGD:

FatalHLA matchAdult onset diseaseSexIntelligence/strengthhttp://www.dnapolicy.org/resources/2006_Hudson_PGD_public_policy_and_public_attitudes.pdf

6Hudson, K. 2006. Preimplantation Genetic Diagnosis: Public Policy and Public Attitudes. Fertility and Sterility 85 (6): 1638-1645.

The PGD process

Egg Sperm

Remove one cellOn day 3rdEmbryo 8 cell stageTest DNA or chromosomesTest resultsHealthy gen conditionsUnhealthy gen conditionsEmbryo implanted on day 4Embryo discardedQuick and rapid technique

Preimplantation Genetic Diagnosis(PGD)

PGD is a state-of-the-art procedure used in conjunction with In Vitro Fertilization (IVF)in which the embryo is tested for certain conditions prior to being placed in the womb of the woman.PGD was first reported in 1990. PGD combines the recent advances in molecular genetics and in assisted reproductive technology

Preimplantation Genetic Diagnosis (PGD) PGD technology involves examination of the chromosomes contained in the polar body, taken from an egg, or a blastomere from a developing embryo to detect and screen genetic abnormality prior to implantation.The original idea arose in the early 1960s when sexing of rabbit embryos at the blastocyst stage was attempted.Twelve years later , The first human pregnancies were obtained from the clinical application of PGD to disorders in couples at risk of having offspring with X linked diseases and cystic fibrosis.Preimplantation genetic diagnosis (PGD) is a state of the art procedure used in conjunction with in vitro fertilization (IVF) to select embryos free of chromosomal abnormalities and specific genetic disorders for transfer to the uterus. These genetic conditions can interfere with embryo implantation, result in pregnancy loss, or in the birth of a child with physical problems, developmental delay or mental retardation

Indications for PGD

Chromosomal DisordersChromosomal rearrangementsInversionsTranslocations Chromosome DeletionsGender determination for severe X-linked diseasesSevere monogenic diseases (cystic fibrosis, thalassaemia, sickle cell anemia, fragile X syndrome, myopathies)Recurrent pregnancy lossAdvanced Maternal Age Couples with >3 IVF failuresEpididymal or Testicular sperm aspiration with >1 IVF failures.

The application of PGD is important in reproductive medicine due to the frequent association between genetic factors and infertility. PGD can be considered in many situations.Some potential parents who carry a chromosomal rearrangement may never achieve a viable pregnancy PGD can be used to determine the sex of the embryo for sex linked disorders It can be recommended to couples at risk of having children with genetic diseases such as cystic fibrosis . As they are healthy carriers of a mutation but do not present symptoms of the disease.Recurrent pregnancy loss shows a high rate of chromosomally abnormal embryosWomen over 35 are more likely to have eggs with extra or missing chromosome (aneuploidy).xand Maternal Age As a woman advances in age, Risk of Aneuploidy is increased.

IVF failureFrequency of chromosomal abnormalities exceeds 55% in individuals with repeated IVF failuresin this group of patients MESA and TESA Studies show a higher incidence of monosomies and trisomies was detected in embryos derived from MESA and TESE72% of chromosomally abnormal embryos is found in this group of patients.

Benefits of PGDIncreased Implantation Rate Reduction in Pregnancy Losses Reduction in the Chance of Having a Child with Aneuploidy

.Reduces the possibility of having to choose to terminate the pregnancy following a diagnosis of a probable genetic disorder.

By replacing only embryos believed to be chromosomally normal there is an increase in implantation rate for those patients at risk In women 35 and over with recurrent pregnancy losses ,,thers is a fifty per cent chance that the chromosomal abnormality of aneuploidy is the cause.More recently, PGD for aneuploidy has been considered a powerful criterion for the selection of viable embryos. Resulting in an increased rate of a successful pregnancy.

How is PGD performed

Day 5/6, Blastocyst biopsy

Methods of PGD/PGSPCR-basedPCR, real-time-PCRQF-PCRSequencing, mini-Sequencing, next-gen sequencingF.I.S.H.array-CGHSNP-array13

PGD TimeLine

19701980199020002010Implementation of the FISH into the cytogeneticsFirst PCR - PGDFISH sex selectionImplementation of the CGH into the cytogeneticsarrayCGH reported for clinical geneticsCGH- PGDaCGH- PGDPCR -PGD for Fresh ETSNP-array PGDFirst delivery after aCGH-PGS

aCGHFirst aCGH-delivery

What? Where? and When?15

PB 1 & 2Blastomere (cleavage stage -day 3)Trophectoderm (blastocyst day 5)

FeaturesPB biopsyBlastomere biopsyTE biopsyIndirect data about the oocyte genotypeMale factor is not taken into accountMosaicism is not excludedDecreasing the embryo viabilitySubsequent self-correction of trisomic embryos is not excludedMore cells = more DNA = more accurate diagnosticsLess mosaicismReduced impact of embryo biopsyEconomic factor: less embryos to be analizedFacilitates the selective embryo transferAllows to modify endometrium if neededAbility to blastocyst cultivation and vitrification are needed

PGD/PGS: Different technologiesCriterionaCGHSNP arrayNext-Gen SequencingQF-PCR/PCRFISHPGSComprehensive chrom. screening+++--Balanced aberrations--?-LimitedNonbalanced abberations+++++MicrodeletionsyndromesLimited++++UPD-++--Single Gene disorders-+/-++-

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Overview For setting up a PGD center Setting up a PGD CentreOrganisation of the PGD CentrePreparation for clinical PGDMisdiagnosisAccreditationExternal quality assessmentESHRE PGD ConsortiumFuture of PGD/PGSWhat makes a good PGD Centre?

Setting up a PGD centre Two ways IVF centre and PGD centre in the same institute preferred Transport PGD

Organisation of the PGD Centre Highly successful IVF unit Patients need genetic and specific PGD counselling Biopsy performed by trained embryologist Diagnosis performed by molecular biologist/cytogeneticist Accredited lab Patient information leaflets and consents Excellent communication between IVF centre and diagnosis lab Join the PGD Consortium

Pretreatment workupFISH Sexing need to check for polymorphisms Translocation protocols developed by cytogeneticist For PGS polymorphic sites PCR Confirmation of mutation on proband and relatives Suitable informative markers to detect contamination Experienced molecular biologist Arrays Depends on if for molecular or cytogenetic Validation of WGA and array Experienced clinical scientist

Workup of diagnosis Validation of method Full authorised report Protocol logged into lab system Prior to cycle internal quality assessment of all reagents and equipment

Clinical cycle Full consultation, information leaflets, relevant consents Need good number oocytes/embryos Patients must not have unprotected sex All cumulus cells removed (maternal contamination) ICSI for all molecular diagnosis (paternal contamination) Medium to support blastocyst growth Clear identification of biopsied cell and embryo number Ensure correct embryo transferred Appropriate witnesses throughout diagnosis Full authorised report logged in PGD and IVF centre

Key points for biopsy/diagnosis lab Counselling Appropriately trained staff Aware of misdiagnosis possibilities Quality control Records ISO/accreditation

Accreditation ISO 15189 Every country has national body How can we help? QM workshop Paper on accreditation of a PGD laboratory, Harper et al, 2010 Establish an accreditation advisory panel Discussion with national accreditation bodies Offer centres help with accreditation process

ISO 15189Management requirements Organization and quality management Quality management system Document control Review of contracts Examination by referral laboratories External services and supplies Advisory services Resolution of complaints Identification of control of non conformities Corrective action/Preventative action Continual improvement Quality and technical records Internal audits Management review

ISO 15189 Technical requirementsPersonnel Accommodation and environmental conditions Laboratory equipment Pre-examination procedures Examination procedures Assuring quality of examination procedures Post-examination procedures Reporting results

What makes a good PGD centre?COMMUNICATIONExcellent IVF PlatformExcellent Diagnostics LaboratoryIntegration of ServicesRigorous Quality Control/Quality AssuranceCommitment to Follow-upComprehensive Ethical ReviewTRANSPORT PGD

Gain genetic information about an embryo or unborn fetus.

Help individuals conceive.

Allow individuals to select embryos based on their genetic makeup.Genetic reproductive technologies can be used to:

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Future of PGDEfforts continue to be focused on improving methods to obtain an accurate diagnosis. PGD holds great promise for the future as techniques and genetic tests are perfected.PGD may become routine in the next few years.

Only a few centers worldwide are offering preimplantation diagnosis. The number of successful cases is small enough such that this method of diagnosis is still considered to be experimental. Efforts continue to be focused on improving methods to obtain an accurate diagnosis from only one or two cells. Techniques are now available to screen for more than one condition simultaneously, however the accuracy of these modifications needs to be tested further. Although there is certainly a demand for this approach, it will continue to be available only in select specialized institutions with advanced in vitro fertilization and molecular biology laboratories. This has tremendous implications for the future of ART. In the same way that ICSI was first performed only in a few centers worldwide and after dissemination of expertise and experience has now become routine in nearly every ART laboratory, embryo biopsy and PGD may become routine in the next few years. This would mean that for a particular individual, if it was found that the majority of her embryos were abnormal for instance, that individual would have powerful objective information that could lead her to seek an oocyte donor, thus sparing the expense and disappointment of repeated ART failures. It would also allow physicians to transfer only normal embryos, which would maximize the chances of a successful outcome irrespective of age. Clearly, this is one of the most exciting developments in the field of reproductive medicine.accurate and sensitive diagnostic techniques, ensuring that ADO is kept to a minimum, testing for all alleles (in the case of couples carrying different mutations), and using DNA markers to identify contamination. Taking this into consideration, the future techniques that may be used for PGD are discussed, such as a whole genome amplification (WGA) and DNA microarrays. accurate and sensitive diagnostic techniques, ensuring that ADO is kept to a minimum, testing for all alleles (in the case of couples carrying different mutations), and using DNA markers to identify contamination.

Its hard to being a good embryo30But even more difficult to detect it