La Diagnosi Genetica Preimpianto (PGD) - Introduction...PGD has also been extended to improve IVF...

C H A P T E RC H A P T E R

16Molecular GeneticsTechniques forPreimplantationGenetic Diagnosis

Francesco Fiorentino, Gayle M Jones

IntroductionHISTORICAL PERSPECTIVE OF PREIMPLANTATIONGENETIC DIAGNOSIS

Couples who are carriers of genetic disorders, includingrecessive or dominant single gene defects, sex-linkedconditions, or chromosome rearrangements, face a repro-ductive risk: affected pregnancies may result in miscarriageor in the birth of a child with significant phenotypicabnormality, sometimes resulting in early death. Suchcouples have the option of undergoing prenatal diagnosisonce a pregnancy is established, either by amniocentesisor chorionic villus sampling (CVS), to allow the detectionof the genetic disorder in the fetus. However, if the fetusis found to be carrying a genetic abnormality the onlyoptions available to couples are to have a child with agenetic disease or to terminate the affected pregnancy.This is a difficult and often traumatic decision, as termi-nation, especially in advanced pregnancies, can havesubstantial psychological and even physical morbidity.Some couples may also experience repeated pregnancyterminations in attempts to conceive a healthy child andmight feel unable to accept further affected pregnancies.The prospect of repeating the process of pregnancy andtermination one or more times in an attempt to achievean unaffected pregnancy will be unacceptable to many.Other couples may not contemplate termination becauseof religious or moral principles.

Such couples have other reproductive choices such asgamete donation or adoption, or to remain childless, buteach of these alternatives has their downsides.

Preimplantation genetic diagnosis (PGD) has beenintroduced as an alternative to prenatal diagnosis in orderto increase the options available for fertile couples whohave a known genetically transmittable disease. PGD is avery early form of prenatal diagnosis. Its intended goal isto significantly reduce a couple’s risk of transmitting agenetic disorder by diagnosing a specific genetic diseasein oocytes or early human embryos that have beencultured in vitro, before a clinical pregnancy has beenestablished. After diagnosis, only embryos diagnosed asunaffected are selected for transfer to the woman’suterus.1,2 The great advantage of PGD over prenatal diag-nosis is that a potential termination of pregnancy isavoided. This gives couples the opportunity to start apregnancy with the knowledge that their child will beunaffected by the genetic disorder. Consequently, PGDdoes not require a decision regarding possible pregnancytermination.

Following its first application in 1990,3 PGD hasbecome an important complement to the presently avail-able approaches for prevention of genetic disorders andan established clinical option in reproductive medicine.The number of centers performing PGD has risen steadily,along with the number of diseases that can be tested4

and new applications and methodologies are introducedregularly. The range of genetic defects which can bediagnosed includes not only single gene disorders but alsostructural chromosomal abnormalities, such as reciprocalor Robertsonian translocations. Avoiding the transfer ofaffected embryos has been shown to not only prevent theconception of babies inheriting most of the common singlegene disorders (SGDs)5-7 and structural chromosome

Author, please provide citation of references 82, 83 in the text

144 A Practical Guide to Setting Up an IVF Lab, Embryo Culture Systems and Running the Unit

rearrangements8 but has also been shown to decrease thenumber of spontaneous abortions associated withstructural chromosome rearrangements.8 The scope ofPGD has also been extended to improve IVF success forinfertile couples, by screening embryos for common orage-related chromosomal aneuploidies in patients who aredeemed to be at increased risk, such as patients ofadvanced maternal age and those having a history ofrepeated miscarriage.9,10

More recently, PGD has been used not only to diagnoseand avoid genetic disorders, but also to screen outembryos carrying a mutation predisposing to cancer or toa late onset disease. Additionally, PGD permits selectionfor certain characteristics, such as HLA matching for tissuetype with the ultimate aim of recovering compatible stemcells from cord blood at birth for transplantation to anexisting sick sibling.11-14

There are several stages during preimplantationdevelopment at which genetic testing can be performed.PGD is usually performed by testing single blastomeresremoved from Day 3 cleavage stage embryos (6–8 cells).An alternative approach is represented by testing the firstpolar body (1PB) before oocyte fertilization (so called pre-conception genetic diagnosis, PCGD)15 or sequentialanalysis of both first and second (2PB) polar bodies(PBs),16 which are by-products of female meiosis asoocytes complete maturation upon fertilization. In womenwho are carriers for a genetic disease, genetic analysis of1PB and 2PB allows the identification of oocytes thatcontain the maternal unaffected gene. Analysis of PBsmight be considered an ethically preferable way to performPGD for couples with moral objections to any micro-manipulation and the potential discard of affected embryos(so called pre-embryonic genetic diagnosis).17 It may alsobe an acceptable alternative for countries in which genetictesting of the embryos is prohibited.15,18 Polar bodyanalysis may also be preferred to blastomere biopsy as itis minimally invasive and potentially less damaging sincethe PB is a discarded by-product of maturation and hasno further role in embryo development leaving the oocyteuncompromised. However, this technique is laborintensive, because all oocytes must be tested despite thefact that a significant number will not fertilize or will fail toform normal embryos suitable for transfer. Furthermore,it cannot be used for conditions where the male partnercarries the genetic disorder, because only the maternalgenetic contribution can be studied.

Single cells for genetic analysis may also be obtainedfrom the embryo at the blastocyst stage of development

on Days 5 or 6 after fertilization.19 Biopsy at this stagehas the advantage of allowing more cells to be sampled(5–10 cells), making genetic tests more robust. It alsoremoves trophectoderm cells, leaving the integrity of theinner cell mass which goes on to form the fetus intact.However, due to the requirement for extended culture ofhuman embryos, together with the fact that same daytesting is required if the blastocyst is to be transferred asa fresh embryo, blastocyst biopsy is currently only usedroutinely in a few centers.

What do We Need to Test for?Currently, there are three principal groups of patients whomay benefit from PGD:1. The first group consists of couples having a reproduc-

tive risk of conceiving a child with a heritable disorder:monogenic disorder (autosomal recessive, autosomaldominant or X-linked disorders) or a chromosomalstructural aberration (such as a balanced translocation).

2. The second group consists of couples that have a poorprognosis to achieve an ongoing pregnancy whoseembryos are screened for chromosome aneuploidies(PGS) to identify euploid embryos for transfer withthe intention of increasing the chances of establishingan ongoing pregnancy. The main indications for PGSare: advanced maternal age, a history of recurrentmiscarriages or repeated unsuccessful implantation andan altered or mosaic karyotype. It has also beenproposed for patients with obstructive azoospermia(OA) and non-obstructive azoospermia (NOA), in casesof unexplained infertility and for patients with aprevious child or pregnancy with a chromosomalabnormality.

3. A third group of indications can be defined as thosethat include the ethically difficult cases that do notnecessarily select against embryos that will definitivelyinherit a genetic disease. This group includes casessuch as HLA typing of the embryo so that the resultantchild can become a cord-blood stem cell donor for asick sibling. Additionally it includes PGD to diagnoseembryos that are capable of resulting in a healthy childwho may in later life be at risk of developing a late-onset disease or have a predisposition to developingcancer. This third group also includes the contentioususe of PGD for sex-selection for social rather thangenetic reasons. A practice that is prohibited in themajority of countries offering PGD.

145Molecular Genetics Techniques for Preimplantation Genetic Diagnosis

PREIMPLANTATION GENETIC DIAGNOSIS FORSINGLE GENE DISORDERS

Many genetic disorders are a consequence of mutationsin single genes. PGD is then indicated for couples at riskof transmitting a monogenic disease to their offspring.

Although it is more than a decade since the first PGDfor Single gene disorder (SGD) was performed,20 thecomplexity of the approach has so far limited its clinicalapplication. PGD is a multidisciplinary procedure thatrequires combined expertise in reproductive medicine andmolecular genetics. Additionally, genetic diagnosis of singlecells is technically demanding, and protocols have to bestringently standardized before clinical application.

Thus only a few centers worldwide are offering PGDfor SGDs as a clinical service. Nowadays, PGD is availablefor a large number of monogenic disorders. It is estimatedthat PGD has been applied for more than 120 differentSGDs in over 4,500 cycles, resulting in the birth of morethan 1,000 unaffected children.4 The monogenic diseasesfor which PGD protocols have been developed generallyreflect those for which prenatal diagnosis is already offeredand includes a continuously growing list of autosomalrecessive, autosomal dominant, and X linked diseases. Themost frequently diagnosed autosomal recessive disordersare cystic fibrosis, -thalassemia, sickle cell disease andspinal muscular atrophy. For autosomal dominantdisorders, the most common indications are myotonicdystrophy and Charcot-Marie-Tooth disease type 1A. PGDfor X-linked diseases has been performed mostly for fragileX syndrome, hemophilia A and Duchenne musculardystrophy.4,8

Almost all genetically inherited conditions that arediagnosed prenatally can also be detected by PGD. It cantheoretically be performed for any genetic disease withan identifiable gene. Diagnostic protocols now exist formore than 200 monogenic disorders.

To establish a diagnostic PGD protocol, extensivepreclinical experiments are carried out on single cells(lymphocytes, fibroblasts, cheek cells or spare blastomeresfrom research embryos), in order to evaluate the efficiencyand reliability of the procedure.

Protocols for genotyping single cells for monogenicdisorders are based on polymerase chain reaction (PCR).Amplified fragments of DNA can then be analyzed accord-ing to the specific requirements of the test; proceduressuch as restriction enzyme digestion, single strandconformation polymorphism (SSCP), denaturing gradientgel electrophoresis (DGGE), allele specific amplification

(ARMS), and recently minisequencing,21 have been usedfor mutation detection. The introduction of fluorescencemultiplex PCR22 allowed the incorporation of linkedpolymorphic markers, to improve the robustness of thePGD protocol or to be used as a tool for indirect mutationanalysis in linkage-based protocols.

Originally, X-linked diseases were avoided by selectionof female embryos, by using the fluorescence in situhybridization (FISH) procedure to identify embryos withtwo X chromosomes.23 A disadvantage of this approachis that half of the discarded male embryos will be healthy,a fact that gives rise to ethical criticism and reduces thechances of pregnancy by depleting the number of embryossuitable for transfer. In addition, half of the female embryostransferred are carriers of the condition. In several X-linkeddominant disorders (e.g. Fragile X syndrome) there is alsothe possibility that, to a varying degree, carrier femalesmay manifest the disease. For many X-linked diseases,the specific genetic defect has now been identified allowinga specific DNA diagnosis. Therefore, there is now aconsensus that it is preferable to use PCR-based tests forsex-linked disorders for which the causative gene is known,instead of performing sex selection.24

Many genetic disorders can now be diagnosed usingDNA from single cells. However, when using PCR in PGD,one is faced with a problem that is non-existent in routinegenetic analysis: namely the minute amounts of availablegenomic DNA. In fact, since PGD is performed on singlecells, PCR has to be adapted and pushed to its physicallimits. This entails a long process of fine-tuning of thePCR conditions in order to optimize and validate the PGDprotocol before clinical application.

There are three main inherent difficulties associatedwith single cell DNA amplification. The limited amount oftemplate makes single-cell PCR very sensitive tocontamination. The presence of extraneous DNA can easilylead to a misdiagnosis in clinical PGD. Cellular DNA fromexcess sperm or maternal cumulus cells that surround theoocyte are a potential source of contamination. These cellscan be sampled accidentally during the biopsy procedure.For these reasons oocytes used for PGD of single genedefects should always be stripped of their cumulus cellsand fertilized by the use of intracytoplasmic sperm injection(ICSI) in which only a single sperm is inserted into theoocyte. Furthermore, any biopsied cells should be washedthrough a series of droplets of medium before transfer tothe PCR tube, and the wash drop should be tested forcontamination.


Other sources of contamination include skin cells fromthe operators performing the IVF/PGD procedure or ‘carryover’ contamination of previous PCR products but thistype of contamination can be minimized or eliminated byfollowing the strict guidelines prescribed by the ESHREPGD taskforce.25

Another problem specific to single-cell PCR is the alleledrop out (ADO) phenomenon.26 It consists of the randomnon-amplification of one of the alleles present in a hetero-zygous sample. ADO seriously compromises the reliabilityof PGD for single gene disorders as a heterozygous embryocould potentially be diagnosed as either homozygousaffected (in which case it would be lost from the cohort ofavailable embryos) or homozygous normal (and, therefore,as suitable for replacement) depending on which allelewould fail to amplify. This is particularly concerning inPGD for autosomal dominant disorders, where ADO ofthe affected allele could lead to the transfer of an affectedembryo.

To obviate the above mentioned problems, PGDprotocols now involve direct mutation(s) detection incombination with analysis of a panel of polymorphic shorttandem repeat (STR) markers that are closely linked tothe gene region containing the disease causing themutation(s) (Fig. 16.1). This approach substantiallyincreases the robustness of the diagnostic procedure anddecreases the possibility of misdiagnosis, providing theadded assurance of a partial ‘fingerprint’ of the embryoand confirming that the amplified fragment is of embryonicorigin. In fact, determination of the specific STR haplotypeassociated with the mutation acts both as a diagnostictool for indirect mutation analysis, providing an additionalconfirmation of the results obtained with the direct geno-typing procedure, and as a control of misdiagnosis due toundetected ADO. Diagnosis is assigned only whenhaplotype profiles, obtained from linked STR markers, andmutation analysis profiles are concordant. The multiplexSTR marker system also provides an additional controlfor contamination with exogenous DNA, as other alleles,differing in size from those of the parents, would bedetected. The experience of a large series of PGD cycles8

strongly suggests that PGD protocols for SGD are notappropriate for clinical practice without including a set oflinked STR markers, consequently this strategy is currentlyused by most PGD laboratories.

One of the most exciting developments in single cellanalysis has been the introduction of protocols designedto amplify the entire genome from a single cell. A recent

important evolution of these whole genome amplification(WGA) protocols is multiple displacement amplification(MDA), a technique that may offer greater accuracy andmore rapid through-put in the future.27 Aliquots of MDAproducts can be taken and used as a source of templatesfor subsequent locus specific PCRs, allowing manyindividual DNA sequences or genes to be analyzed in thesame cell. The main limitations of using this method onsingle cells are the high ADO and preferential amplificationrates compared with direct PCR on DNA from a singlecell.27,28 Moreover WGA provides a sufficient supply ofsample DNA available for other applications, such asaneuploidy screening by using array-CGH technique. Itcould therefore be envisaged that in the future everyembryo tested for a monogenic disorder is also routinelyscreened for aneuploidy, investigating all chromosomes.

PGD FOR CHROMOSOME TRANSLOCATION

Individuals who carry a balanced chromosomal trans-location (reciprocal or Robertsonian) typically suffer nooutward manifestations of the rearrangement. Thetranslocations, however, are associated with the productionof large numbers of gametes with an unbalanced chromo-some make-up due to excess or missing geneticmaterial.8,29,30 These unbalanced gametes lead to a greaterchance of the patient being infertile and/or at high risk ofconceiving chromosomally abnormal pregnancies that leadto recurrent spontaneous abortions or children withcongenital anomalies and mental retardation.29

PGD has been offered to carriers of balancedtranslocations as an alternative to prenatal diagnosis andtermination of unbalanced pregnancies.8,31-33 Previousstudies have shown that PGD for translocations has thepotential to improve livebirth rates by either reducing therisk of recurrent spontaneous abortions, minimize the riskof conceiving a chromosomally abnormal baby, or toimprove pregnancy rates in infertile couples, e.g. afterfailed IVF attempts.8,9,29,31,32,34 Application of PGD tocarriers of balanced translocations can decrease the riskof these adverse outcomes by selecting for transfer onlythose embryos with a normal/balanced chromosomalcomplement.30,32,34

Fluorescence in-situ hybridization (FISH) is, to date,the most widely used method for detecting unbalancedchromosome rearrangements in embryos.4 This techniqueuses DNA probes labeled with distinctly coloredfluorochromes that bind to specific DNA sequences uniqueto each chromosome. Imaging systems enable the


Fig. 16.1: Preimplantation genetic diagnosis (PGD) for -thalassemia. Pedigree of a couple carrying -thalassemia mutationsand examples of different results of the HBB gene mutation analysis. Informative STR markers are ordered from telomere (top)to centromere (bottom). The numbers in STR markers represent the size of PCR products in base pairs. STR alleles linked tothe paternal and maternal mutations are colored in red. Embryo 1 is a carrier for IVSI-110 G>A mutation; Embryos 2 and 7are carriers for IVSII-745 C>G mutation; Embryos 3 and 6 are normal; Embryo 8 is a compound heterozygote for the twomutations. Embryo 5 is also affected, although mutation analysis result shows a heterozygosity for mutation IVSI-110 G-A. Infact, linked STR markers highlight an allele drop-out (ADO) of the IVSII-745 C>G mutation

fluorescent probe signals to be identified and counted todetect missing or excess chromosomal material.

A commonly used FISH strategy for detection of theabnormal segregation in reciprocal and Robertsoniantranslocations involves the use of commercially availablecentromeric, locus specific and subtelomere probes(Fig. 16.2), allowing for a simplified approach thatlaboratories can apply routinely.8,29,31

Analysis of reciprocal translocations for PGD is difficultsince each translocation is effectively unique to the familyor person within which it occurs, and the breakpoint mayhave arisen at any point on any chromosome, thusdifferent combinations of FISH probes are usually requiredfor each couple. FISH strategies for assessment ofreciprocal translocations use three differentially labeledprobes, two probes that are specific for the subtelomericregions of the translocated segments, combined with a

centromeric probe.5,29 Analysis of Robertsonian trans-locations is simpler, involving the use of specific probeschosen to bind at any point on the long arm of eachchromosome that is involved in the translocation. Theabove combination of probes allows embryos that carryan unbalanced chromosome complement to bedistinguished from healthy ones. However, both strategiesdo not discriminate between non-carrier embryos andthose that carry the balanced form of the translocation.

Although relatively successful, the FISH procedure istechnically demanding and harbors several technicallimitations that are well-documented9,35,36 and includehybridization failure (lack of FISH signals), signal overlap,signal splitting and poor probe hybridization, as well asproblems related with the fixation process, such as cellloss and variable cell fixation. Since FISH was firstintroduced in clinical diagnosis, improvements have been


Fig. 16.2: Results obtained from a PGD case for Robertsonian translocation13,14 (Right) Capillary electrophoresis of fluorescentPCR products, after multiplex amplification of a set of polymorphic STR markers located along chromosome 13. On top of theelectropherogram the marker name is located above the corresponding alleles (peaks). A normal diploid embryo (C) has thenormal complement of each parental chromosome, thus two alleles of a chromosome specific STR are determined as twopeaks. Embryos with a normal copy number for a given chromosome will show a heterozygous pattern for all the STRs used.The observation of an extra STR allele as a three peak pattern is diagnostic of the presence of an additional sequence whichrepresents an additional chromosome, as in the case of a trisomy. Trisomic embryos will produce trisomic patterns for allmarkers on the same chromosome (B). The observation of only one STR allele as a one peak pattern is diagnostic of themissing of the sequence from one chromosome, as in the case of a monosomy. Monosomic embryos will show a homozygotepattern for all the STRs used for a given chromosome (A). (Left) Fluorescence in situ hybridization (FISH) results from the samePGD case as above. (Center) Graphical representation of chromosomes 13, 14, and the STR markers used


established to diminish the error rate of the tech-nique.37-39 Technical skill and sound laboratory practicescan minimize most of the limitations of FISH-basedmethods,40 but certain shortcomings remain. Interpretationerrors due to the technical issues described above canaffect the accuracy of the interpretation of the results,leading to misdiagnosis of embryos both in eliminatingsuitable (normal/balanced) embryos for transfer, or worse,including abnormal embryos in the transfer cohorterrantly.41 Error rates of FISH protocols for translocationhave been reported in some studies to range from 0 to10%, with an average error rate of 6%.8,35,42

Increasingly, new techniques for chromosome analysisin embryos are being sought in an attempt to improve oncurrent FISH test method performance. Recently, a poly-merase chain reaction (PCR)-based PGD approach fordetection of chromosomal imbalances on embryos derivedfrom both reciprocal and Robertsonian translocationcarriers was proposed as a valuable alternative to the FISH-based PGD protocols.43 The approach consisted of afluorescent multiplex PCR using short tandem repeat (STR)markers flanking the translocation breakpoints on bothchromosomes involved, which tracks al l meioticsegregations (Fig. 16.2). This approach aims to overcomeseveral of the above listed limitations related with theFISH technique while providing significant improvementsin terms of test performance, automation, turnaroundtime, cost effectiveness, sensitivity and reliability of theinformation obtained.

It is relatively common for PGD of chromosomaltranslocations to be combined with aneuploidy screening,to assess common aneuploidies for patients of advancedmaternal age. This can be done using both FISH andPCR-based protocols, but involves aneuploidy screeningof only a limited number of chromosomes and focuses onthe chromosomes most often found to be aneuploid inprenatal samples or material from miscarriages.44-46 Thismay result in the transfer of reproductively incompetentembryos with aneuploidy for chromosomes not analyzed.

Comprehensive chromosome screening techniques,such as array comparative genomic hybridization (array-CGH),47-49 have also been introduced into current routinePGD laboratory practices recently. Microarrays used forarray-CGH are made up of DNA sequences specific tohuman chromosomes spotted onto a platform, usually aglass slide. Array-CGH enables the assessment of all thechromosomes by comparing the studied DNA with anormal sample and is appropriate for analysis of a single

copy of DNA as for a single cell.47 This method has ahigh-resolution and is amenable to automation.

Some of the most promising progress toward develop-ing a comprehensive 24 chromosome analysis methodhas been made possible through the combination of wholegenome amplification (WGA), a protocol able to amplifythe entire genome from a single cell, and array-CGH.47,50,51 The array-CGH procedure involves screeningof the entire chromosome complement, rather than thelimited chromosome assessment typically used for thepurpose of preimplantation genetic screening (PGS). Thearray-CGH procedure detects imbalance across thegenome allowing identification of whole-chromosomeaneuploidies and even small structural aberrations. Thistechnique, applied to embryos derived from translocationcarriers, would offer a complete analysis of the embryoby providing information regarding not only thechromosomes involved in the translocation, but also theploidy of all 24 chromosomes (Fig. 16.3).52

PREIMPLANTATION GENETIC SCREENING FORCHROMOSOME ANEUPLOIDY

Selection of the most competent embryo(s) for transfer isgenerally based on morphological criteria. However, manywomen fail to achieve a pregnancy after transfer of goodquality embryos. One of the presumed causes is that suchmorphologically normal embryos show an abnormalnumber of chromosomes (aneuploidies).53 Aneuploidembryos have a lower survival rate than normal embryos(22 pairs of autosomes and 2 sex chromosomes) and themajority fail to implant.

Aneuploidy screening of embryos derived fromsubfertile patients undergoing IVF is probably the mostfrequent indication for PGD.4 Preimplantation geneticdiagnosis for aneuploidy screening, also named preimplan-tation genetic screening (PGS), enables the assessment ofthe numerical chromosomal constitution of cleavage stageembryos. It aims to identify and select for transfer onlychromosomally normal (euploid) embryos in order toincrease the implantation and pregnancy rate for IVFpatients, lower the risk for miscarriage and reduce the riskof having a baby with an aneuploidy condition.54

PGS and PGD are often presented as similar treat-ments, although they have completely different indications:PGS aims to improve pregnancy rates in subfertile couplesundergoing IVF/ICSI treatment whereas PGD aims toprevent the birth of affected children in fertile couples witha high risk of transmitting genetic disorders.


The main indications suggested for PGS are repeatedimplantation failure,55 advanced maternal age,54-56

repeated miscarriage in patients with normal karyo-types,10,57,58 an altered/mosaic karyotype in one or bothof the prospective parents59,60 and severe male factorinfertility.61,62

Limitations of PGS

The detection of chromosomes in a single biopsiedblastomere has usually been achieved using FISH.Essentially, FISH probes to detect those aneuploidies mostcommonly observed after birth or in miscarriages (involv-ing detection of chromosomes X, Y, 13, 16, 18, 21, and22) are used.63 This panel of probes has the potential ofdetecting over 70% of the aneuploidies found in sponta-neous abortions.64 Aneuploidy conditions (involvingchromosomes 8, 9, 15, and 17) that cause lack of implan-tation or can result in a miscarriage early in pregnancyhave also been tested.57,63

However, FISH-based PGS allows identification ofaneuploidies only for a limited number of chromosomes(5, 9, or 12 chromosomes).44-46 Commercial FISH probepanels are available that will identify 5-6 chromosomes ata time by different colors (fluorochromes). The number ofchromosomes that can be tested at any one time is limitedby the range of fluorochromes available and the eye’s

ability to distinguish between different colors. The numbersof chromosomes investigated may be increased by washingoff the first panel of probes and re-hybridizing with anadditional panel of probes but this increases the time takento arrive at a diagnosis. Re-hybridization can only beperformed, with any clinical accuracy, once, and thistherefore limits the number of chromosomes that can beanalyzed. Several studies have demonstrated thataneuploidies may involve all 24 chromosomes.49,65-67

Therefore FISH analysis may result in the transfer ofreproductively incompetent embryos with aneuploidypresent in the chromosomes not analyzed.

PGS currently has several further disadvantages thatlimit its clinical value. The main concern is the elevatedmosaicism rate observed in the human cleavage stageembryo. Mosaicism is defined as the embryo having cellswith different chromosome make-up and it has been foundin up to 57% of Day 3 biopsied embryos.68,69 Mosaicismmay represent a major source of misdiagnosis (60%)because of both false-positive and false-negative results.

Besides mosaicism, several technical limitationsinherent to the FISH technique have been described. FISHis considered to have an error rate between 5 and10%.54,63 Overlapping signals may be a source of misdiag-nosis resulting in false diagnosis of monosomies. Signalsplitting has also been described, resulting in the detection

Fig. 16.3: Examples of array-CGH based preimplantation genetic diagnosis results from a chromosomally unbalanced embryoderived from a patient carrying a balanced translocation 46,XY,t(5;8)(q31.1;q22.1), consistent with meiotic adjacent-1 segregation.The imbalances include a partial trisomy 5q31.1-qter, detected as a shift of the clones located in the above region towardsthe green line (gain), and a partial monosomy 8q22.1-qter, identified with a shift of the specific clones towards the red line(loss)


of false trisomies. Finally, evidence suggests that up tohalf of all embryos identified as aneuploid at the cleavagestage and that survive to the blastocyst stage will selfcorrect,42,70 therefore an abnormal result may notnecessarily indicate that the embryo is abnormal and ill-fated.

As a consequence of the above limitations, it has beenquestioned whether some normal embryos might beexcluded from the cohort that is considered suitable forembryo transfer, which, especially in older women whomight have small embryo cohorts, could result in the failureto reach embryo transfer.

The Debate on PGS Usefulness

In the last years, there has been a steady increase in thenumber of PGS cycles reported to the ESHRE PGDConsortium, from 116 cycles in the data collection from1997-1998 to 1722 cycles in 2003.4 The rapid increase inthe use of this procedure has raised questions about itsefficacy for routine use.

Since the publication of the first articles on PGS usingcleavage-stage embryos71 and polar bodies,72-74 therehave been a number of non-randomized comparativestudies of IVF/ICSI with or without PGS, for advancedmaternal age or repeated implantation failure. Most ofthese studies reported that PGS increases the implantationrate9,10,55,56 decreases the abortion rate and reducestrisomic conceptions.37,57,75

Three randomized controlled PGS trials have beenperformed for advance maternal age76-78 and the resultsindicate that PGS does not improve ongoing pregnancyor livebirth rates. In contrast, one of these studies showedthat PGS decreased the chance of achieving an ongoingpregnancy or livebirth.77 Furthermore, a randomizedcontrolled trial (RCT) evaluating the effectiveness of PGSin women under the age of 35 undergoing IVF treatmentwith single embryo transfer did not show a benefit forPGS.79

The two largest randomized trials for advancedmaternal age have been criticized.80,81 Staessen andcollaborators76 were criticized for biopsying two cellsinstead of one cell, which may have a negative impact onembryo survival.38 The study by Mastenbroek andcollaborators77 was criticized for: its high percentage ofembryos without a diagnosis (20%), not including probesfor chromosomes 15 and 22 and the low ongoingpregnancy rate in the control group (39 pregnancies/195oocyte retrievals, 20%).38,84

There are now 11 RCTs published on PGS, 10 usingcleavage-stage biopsy76-78,85-91 and one using blastocystbiopsy.45 All have used FISH testing of a limited numberof chromosomes and none have shown an improvementin delivery rates, with some showing a significant decreasein delivery rates after PGS. Most of the RCTs have beenfor patients with advance maternal age.76-78,87,89,91

The above mentioned RCTs have shown that PGS, asit was practiced, has not provided the expected benefits.There are many possible reasons why these clinical studiesfailed to deliver the expected improvements in IVFoutcome. Putative explanations for this poor clinicalperformance could be attributed to an incomplete under-standing of important aspects of embryo biology, such asembryonic chromosomal mosaicism, that is present onDay 3 of development (i.e. the tested blastomere is notrepresentative of the whole embryo),92 or self-correctionof aneuploidy within the embryo, which may decreasethe chances of a livebirth by prematurely labeling anembryo as abnormal. Indeed, high levels of chromosomalmosaicism have been observed in blastomeres fromcleavage-stage embryos.66,72,73,93

Alternatively, it has been argued that inadequatecytogenetic methods may have led to reduced diagnosticaccuracy and elimination of any potential benefit ofscreening.94 In fact, all prior clinical trials using PGS forIVF have used FISH, which screens for a minority ofchromosomes (up to 12 chromosomes).37 The majorityof FISH-based methods focus on the chromosomes mostoften found to be aneuploid in prenatal samples or materialfrom miscarriages. However, these chromosomes are notnecessarily the most relevant for early embryos. Thus, thelimitations of the FISH technology could have compro-mised the results of these studies. Therefore, future workin this area should explore the use of new technology thatallows for more comprehensive screening of chromosomes(array-based technology).

Recently, microarray-based PGS approaches for all 24chromosomes have been proposed in order to overcomethe technical difficulties that beset earlier PGS studies,allowing screening of the entire chromosome complement,rather than the limited chromosome assessment achievableby FISH49,67 (Fig. 16.4).

Data from comprehensive aneuploidy screeningshowed that aneuploidies may occur in preimplantationembryos in any of the 24 chromosomes, indicating thataneuploidy screening of all chromosomes is necessary todetermine whether an embryo is chromosomally


normal.49,67 However, screening for all 24 chromosomesin a single blastomere biopsied from a cleavage stageembryo may still be compromised by the degree ofmosaicism that has been observed in such embryos.Aneuploidy detection in PB1 and PB2 will detect themajority of the aneuploidies that arise from the maternalgenome, which is particularly relevant for patients ofadvanced maternal age, but this technique ignores thepossible contribution from the paternal genome and anyaneuploidies that may arise during aberrant mitoticdivision. Trophectoderm biopsy at the blastocyst stage ofdevelopment is another alternative but too few studieshave been undertaken to evaluate if this is a more accuratereflection of the chromosomal status of the inner cell massand ultimately the developing fetus.

The debate on the usefulness of PGS is still ongoing.Further data are required to establish whether PGS resultsin enhanced livebirth rate, and if this is the case, to identifywhich patients may benefit. The only effective way toresolve this debate is to perform well-designed and well-executed randomized controlled clinical trials. The designof further studies should include an adequate randomi-zation protocol with a clear stratification concerning theindications to perform PGS, the replacement of the samenumber of embryos in both study and control groups andthe healthy livebirth rate per treatment cycle as the mainoutcome measure. Additional points of interest for futureresearch are the implementation of new technologies forchromosome analysis, e.g. array comparative genomichybridization, that enable a complete assessment of the

numerical chromosomal constitution of preimplantationembryos.

ETHICALLY DIFFICULT INDICATIONS

The proposed indications for use of PGD are beingextended. New uses include PGD to detect mutations forsusceptibility to cancer and for late onset disorders. Inaddition, parents with children needing hematopoietic stemcell transplants have used PGD to ensure that their nextchild is not only free of disease but also provide a goodtissue match for an existing sick child.

PGD for Late Onset Disorders

One of the proposed uses of PGD is the identification ofembryos at risk for late-onset or adult-onset diseases.61,62

Many serious genetic disorders, such as Alzheimer’s diseaseor Huntington disease, have their onset later in adult life,with the result that those affected remain healthy for yearsor even decades, until the onset of the disease, living anormal healthy life. However, some of these disorders areprogressive and disabling, or even lethal, and account forserious ill health. On the other hand, the high probabilityor certainty of developing the disorders, and their incurablenature, can lead to a stressful life as the patient waits forthe first symptoms to occur and anticipates prematuredeath.

Thus late onset disorders present a dilemma: at birtha child will be healthy and free of disease, but will carrythe potential to develop ill health in later life. Ethically,

Fig. 16.4: Examples of array-CGH based preimplantation genetic screening (PGS) results from day-3 biopsy.The embryo shows aneuploidy of chromosomes 12, 17, 21 (loss) and 18 (gain)


the question is whether the burden of carrying susceptibilitygenes is so great for the child and parents that the burdensof IVF and PGD to screen embryos to avoid the affectedchildren are justified. Many believe PGD is ethicallyacceptable for these indications because of the heavyburden imposed on patients who are carriers of this typeof disease.

The use of PGD for asymptomatic individuals withthe Huntington mutation is one of the most commonapplications of PGD for late onset disorders.4 “Non-disclosure” PGD for Huntington’s disease is applied inthose cases in which the prospective parent at risk doesnot wish to be informed about his or her own carrier statusbut wants to have offspring free of the disease.95 Embryoscan be tested for the presence of the mutation withoutrevealing any of the details of the cycle or diagnosis tothe prospective parents. Non-disclosure testing is contro-versial and not generally endorsed by professionals,96,97

because it puts practitioners in an ethically difficultposition, i.e. when no embryos are available for transferand a mock transfer has to be carried out to avoid the

patient suspecting that he/she is a carrier or having toundertake PGD cycles even when the results of previouscycles preclude the patient being a carrier.

The ESHRE ethics task force24 currently discouragesnon-disclosure testing, recommending the use of exclusiontesting instead. Exclusion testing is based on a linkageanalysis with polymorphic markers, in which the parentaland grandparental origin of the chromosomes can beestablished (Fig. 16.5). In this way, only embryos arereplaced that do not contain the chromosome derived fromthe affected grandparent, avoiding the need to detect themutation itself.96

However, exclusion testing PGD is also considered asethically dubious by some because embryos with an allelefrom an affected grandparent will be excluded for transfer,even though in only half of the cases the allele will beaffected.

PGD for Inherited Cancer Predisposition Syndromes

Inherited cancer predisposition has become one of theemerging indications for PGD.98 The use of PGD to screen

Fig. 16.5: Preimplantation genetic diagnosis (PGD) for Huntington disease (HD) by exclusion testing. Familial pedigree froma couple at risk for HD. Informative STR markers, linked to the HD gene, are ordered from telomere (top) to centromere(bottom). The numbers in STR markers represent the size of PCR products in base pairs. A and B are at risk haplotypes,inherited from the grandparent affected by HD. Embryos 2 and 4 are normal; Embryos 1 and 3 are at risk for HD


out embryos carrying a mutation predisposing to cancer(e.g. breast/ovarian cancer – BRCA1 and BRCA2 genes;Li-Fraumeni syndrome–p53 gene; neurofibromatosis–NF1and NF2 genes; retinoblastoma–RB1 gene; familialadenomatous polyposis – APC gene; hereditary non-polyposis colon cancer – MSH2 and MLH1 genes; vonHippel-Lindau syndrome – VHL gene), prevents the birthof children who would face a greatly increased lifetimerisk of cancer, and hence require close monitoring,prophylactic surgery, or other preventive measures.Through PGD, couples with a familial history of cancer,where one partner has the high risk gene, now have anopportunity to start a pregnancy knowing that theiroffspring will not carry the cancer-predisposing mutation.

Contrary to the monogenic late-onset diseases, whichhave full penetrance (i.e. a person found to have amutation will inevitably get the symptoms in the future),having a predisposing mutation for a susceptibility geneonly increases the risk of developing the disease (e.g.inheritance of a familial breast cancer mutation in theBRCA1 gene is associated with an 80% life time risk ofbreast cancer and other BRCA1 related cancers), since itseffect is modified by other genes and other factors. Forthe hereditary forms of breast cancer (due to mutations inBRCA1 and BRCA2 genes) and for some hereditary formsof colorectal cancer (e.g. FAP and HNPCC genes) theabsolute risk figures are relatively high but, unlike late-onset diseases, the above cancer predisposition syndromescan usually be treated or preventive measures adopted.

As couples present for PGD for heritable cancer genes,there is likely to be widespread debate in the communityabout the associated ethical issues, including disposal ofembryos carrying the cancer-predisposition gene mutationand the practice of eugenics.99 Although these indicationsdo not involve diseases that manifest themselves in infancyor childhood, the conditions in question lead to substantialhealth problems for offspring in their thirties or forties.However, owing to the adult onset of hereditary cancer,prenatal diagnosis raises ethical issues surrounding theacceptability of terminating an affected pregnancy.

PGD for HLA Matching

PGD of single gene disorders, combined with humanleukocyte antigen (HLA) matching, represents one of themost recent applications of the technique in reproductivemedicine.11-14 This strategy has emerged as a tool forcouples at risk of transmitting a genetic disease to allow

them to select unaffected embryos that are HLA tissuetype compatible with those of an existing affected child.In such cases, PGD is used not only to avoid the birth ofaffected children, but also to conceive healthy childrenwho may also be potential HLA-identical donors ofhematopoietic stem cells (HSC) for transplantation tosiblings with a life-threatening disorder. At delivery, HSCfrom the newborn umbilical cord blood can be collectedand used for the hematopoietic reconstruction of theaffected sibling.

At present, allogenic HSC transplantation representsthe only curative treatment for restoring normal hemato-poiesis in severe cases of neoplastic (e.g. leukemia) orcongenital (e.g. -thalassemia) disorders affecting thehematopoietic and/or the immune system. A criticalfactor associated with favorable outcome in stem celltransplantation is the use of HLA-genotype identicaldonors, and HLA identical siblings provide the bestchance to the recipient in the achievement of a successfultransplantation. Unfortunately, because of the limitedavailability of HLA-matched sibling donors, most of thepatients face the option of transplantation using a volunteerunrelated matched donor, identified from national orinternational registers. In these cases, the results are lessfavorable compared to the matched-sibling transplant.HLA mismatches are increased using unrelated donors,with a consequent higher incidence of both transplant-related mortality and graft-versus-host disease.

Therefore, if no HLA identical donor is available inthe family, an increasing number of couples with a childaffected by a hematopoietic disorder are considering theuse of IVF and PGD techniques for therapeutic purposes.Before the existence of PGD, natural conception followedby prenatal diagnosis and possible termination of preg-nancy was the only option when trying to find an HLAmatched future sibling.100

Verlinsky and collaborators11 described the first pre-implantation HLA matching case in 2001. Since then, HLAtyping has become an important PGD indication in fourcountries: USA,11 Italy,12,14 Turkey,101,102 and Belgium.13

The procedure is particularly indicated for patients withchildren affected by Fanconi anemia, -thalassemia, sicklecell anemia, Wiscott-Aldrich syndrome (WAS),X-linked adrenoleukodystrophy (X-ALD), X-linked hyper-IgM syndrome (HIGM), X-linked hypohidrotic ectodermaldysplasia with immune deficiency (HED-ID) and othersimilar disorders, that require a HLA-compatible HSC orbone marrow donor to be effectively treated. The great


difficulty in finding a HLA-matched donor, even amongfamily members, led to the application of preimplantationHLA matching also for diseases such as acute lymphoidleukemia (ALL), acute myeloid leukemia (AML), orsporadic Diamond-Blackfan anemia (DBA).14 For theseconditions, not involving testing of a causative gene, PGDfor HLA matching becomes the primary indication.

Technically, PGD for HLA typing is a difficult proceduredue to the extreme polymorphism of the HLA region.Taking into account the complexity of the region (presenceof a large number of loci and alleles) the use of a directHLA typing approach would require standardization of aPCR protocol specific for each family, presenting differentHLA allele combinations, making it time consuming andunfeasible. The use of a preimplantation HLA matchingprotocol irrespective of the specific genotypes involvedmakes the procedure more straightforward. Currently, PGDlabs use a strategy based on a flexible indirect HLA typingprotocol applicable to a wide spectrum of possible HLAgenotypes.11-14 The approach involves testing of singleblastomeres by fluorescent multiplex PCR analysis ofpolymorphic STR markers, scattered throughout the HLAcomplex, obtaining a fingerprint of the entire HLA region(Fig. 16.6).

Although several publications11-14 on the argumenthave demonstrated the success and the usefulness of thePGD/HLA approach, some limitations have to be consi-dered. Firstly, advanced maternal age as well as a poorovarian response to hormonal hyperstimulation are knownto have a major impact on the number of retrievableoocytes and consequently, on the number of embryosavailable for analysis, reducing the likelihood of findingtransferable embryos. Thus several IVF cycles may benecessary to obtain a pregnancy and a livebirth. Secondly,the selection of a donor embryo for HSC transplantationbefore implantation is restricted by the intrinsic geneticconstitution of the embryos: only 1/4 or 25% (HLA typing),3/16 or 19% (HLA typing and mutation analysis) ofembryos will be transferable. These numbers can be furtherreduced by any requirement to ensure that the embryo isalso disease free. Thirdly, the clinical success rate of PGD/HLA typing is low. This is a common phenomenon in allcenters offering HLA/PGD and couples should becounseled for this because they have put all their hope inthis approach to cure their sick child.

Ethical discussion concerning PGD in combination withHLA is ongoing, mainly because the procedure involvesembryo selection.100 In the case of embryo selection based

on HLA typing alone the ethical discussion is even strongersince the selection here is based on a non-disease traitand opponents claim that the child to be born is instru-mentalized, although some authors maintain that theKantian imperative is not breached since the future donorchild will not only be a donor but also become a lovedindividual within the family. In the case that there was anexisting HLA-matched sibling, we would find it acceptableto use that child as a donor of hematopoietic stem cells.Unless the use as a donor would be the sole reason forcreating the child, there would be no violation of itsautonomy.

The question of the motivation of the couples alsomay raise concerns. They could be tempted to have anadditional child solely for the purpose of furthering theinterest of the existing sibling and not because they desireanother baby. This difficult ethical issue can be partiallyaddressed by careful genetic and psychological counselingof the couples to ascertain their real motivation. However,considering the efforts by the parents to save their sickchild, it is very unlikely that they would not treat the saviorchild as equal to the existing child.

Finally, ethical questions are raised by the considerablenumbers of embryos that need to be generated in orderto identify one that is disease free and closely matchedwith the sick sibling. However these questions can bemitigated by the fact that all embryos unaffected by thedisease but non-HLA matched can be cryopreserved forfuture use, in the event that the couples wish to have morechildren unaffected with the disease.

Overall, most consider the above criticisms a minorconcern when compared with the possibility of saving achild’s life from a devastating disease.

ConclusionPGD is an effective clinical tool for assisted reproductionand genetic screening. From the patients’ perspective, PGDis an important alternative to standard prenatal diagnosis.Low pregnancy and birth rates, and the high cost of theprocedure, however, make it unlikely that PGD willcompletely supersede the more conventional methods ofprenatal testing. PGD remains a complex combination ofdifferent technologies, which involve reproductivemedicine as well as clinical and molecular genetics andrequire the close collaboration of a team of specialists.Rapid advances in molecular genetics are likely to stimulatefurther use of PGD and to encourage a substantial increase


Fig. 16.6: Preimplantation HLA matching in combination with PGD for Wiskott-Aldrich syndrome (WAS), resulting in the birthof a carrier female, HLA matched with the affected sibling. Upper pane: Determination of the different haplotypes from father,mother and affected child (lower panel-black square) by segregation analysis of the alleles obtained after STR genotyping ofthe WAS gene. Informative STR markers used are ordered from telomere (top) to centromere (bottom). Paternally and maternallyderived HLA haplotypes, matched to the affected child, and STR alleles linked to the paternal and maternal mutations, areshown in boldface. Examples of different results of WAS gene mutation analysis and HLA haplotyping from biopsied blastomeresare shown in the lower panel. Embryos 5 (normal male) and 11 (carrier female) was diagnosed as HLA non-identical. Embryo18 was diagnosed as HLA matched, but affected. Embryo 14, representing a carrier female was diagnosed as HLA matchedwith the affected sibling and was transferred, resulting in the birth of an HLA matched unaffected child. (ET = EmbryoTransfer)

in the range of genetic conditions for which PGD is offered.The accuracy of procedures will be improved and itsclinical application will be simplified. In the future, PGDwill play an increasing role as a specialized clinical proce-

dure, becoming a useful option for many more coupleswith a high risk of transmitting a genetic disease, to preventthe birth of affected children, and for infertile couples toimprove IVF success rates.


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La Diagnosi Genetica Preimpianto (PGD) - Introduction...PGD has also been extended to improve IVF...

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