A methodological overview on molecular preimplantation geneticdiagnosis and screening: a genomic future?
Xavier Vendrell1∗ and Rosa Bautista-Llácer2
1Reproductive Genetics Unit, 2Molecular-PGD Laboratory, Sistemas Genómicos S.L., Paterna, Spain
The genetic diagnosis and screening of preimplantationembryos generated by assisted reproduction technology hasbeen consolidated in the prenatal care framework. The rapidevolution of DNA technologies is tending to molecularapproaches. Our intention is to present a detailed methodo-logical view, showing different diagnostic strategies based onmolecular techniques that are currently applied in preimplanta-tion genetic diagnosis. The amount of DNA from one single, ora few cells, obtained by embryo biopsy is a limiting factor forthe molecular analysis. In this sense, genetic laboratorieshave developed molecular protocols considering this restrictivecondition. Nevertheless, the development of whole-genomeamplification methods has allowed preimplantation geneticdiagnosis for two or more indications simultaneously, like theselection of histocompatible embryos plus detection of mono-genic diseases or aneuploidies. Moreover, molecular tech-niques have permitted preimplantation genetic screening toprogress, by implementing microarray-based comparative gen-ome hybridization. Finally, a future view of the embryo-genetics field based on molecular advances is proposed. Thenormalization, cost-effectiveness analysis, and new technologi-cal tools are the next topics for preimplantation genetic diag-nosis and screening. Concomitantly, these additions toassisted reproduction technologies could have a positive effecton the schedules of preimplantation studies.
Couples with a known genetic risk should start their repro-ductive project with accurate preconception genetic counsel-ling. From the prevention point of view, all the informationconcerning different reproductive options should beexposed, ensuring that patients understand what is beingexplained. However, the rapid evolution of methodologicalapproaches in genetic diagnosis makes this ideal situationdifficult, particularly in the assisted reproductive field. Inthis context, the preimplantation genetic diagnosis (PGD)and preimplantation genetic screening (PGS) underwent afast advance very recently. PGD was proposed as a very earlyform of genetic diagnosis in 1990 [Handyside et al. 1990]with the purpose of avoiding the transmission of genetic dis-eases to offspring. Nowadays, PGD represents a well-established alternative to prenatal diagnosis and terminationof pregnancy, in couples at risk of transmitting disorders.
Since the first PGD application, it has been made avail-able for a large number of rare genetic disorders [Preimplan-tation Genetic Diagnosis International Society 2008; Harperet al. 2012] and the number of cycles increases year by year(Harper et al. 2010b). The implementation of new technol-ogies to PGD increases the diagnostic capacity and extendsthe application of PGD, not only for the most commonmonogenic diseases [Fiorentino et al. 2006], but also forscreening aneuploidies on in vitro fertilization (IVF) patients(advanced maternal age or recurrent miscarriage factor)[Verlinsky et al. 1999; Munnè et al. 2002; Rubio et al.2003]. Nevertheless, this last application has recently beensubjected to a deep and interesting debate, reorienting theinitial methodology [Harper et al. 2010a]. Also, this has per-mitted a new systematic analysis of the PGD indication[Harper and Harton 2010]. Moreover, PGD is also appliedto more complex cases like single gene disorders combinedwith fluorescent in situ hybridization (FISH)-based aneu-ploidy studies [Verlinsky 2006] or human leukocyteantigen (HLA) matching [Verlinsky et al. 2001; Fiorentinoet al. 2004; Fiorentino et al. 2005]. PGD has even beenapplied to HLA haplotyping combined with aneuploidy
∗Address correspondence to Xavier Vendrell PhD, Head of the Reproductive Genetics Unit, Sistemas Genómicos S.L., Ronda G. Marconi 6, ParqueTecnológico de Valencia. 46980 Paterna, (Valencia) Spain. E-mail: [email protected]
testing [Rechitsky 2006]. The combination of various PCR-based analyses on single cells, has permitted two indicationssimultaneously, for example testing two single genedisorders in the same couple [Altarescu et al. 2007; Alberolaet al. 2009]. In addition, the recent application of microarraytechnologies in this field is quickly changing the diagnosticstrategies. This new approach allows combining single genePGD with aneuploidy screening of 23 autosomes andsexual chromosomes, simultaneously [Brezina et al. 2011].Recently, there has been a clear tendency to perform molecu-lar techniques. In this sense, aneuploidy screening and chro-mosomal rearrangement studies are quickly being replacedby molecular biology approaches based on microarrayplatforms (reviewed by [Harper and Harton 2010]), butalso based on the analysis of short tandem repeat (STR)haplotypes [Fiorentino et al. 2010; Traversa et al. 2010].
In this fast-evolving scenario, a comprehensive review hasbeen published recently [Harper and SenGupta 2012]revisiting the historical evolution of PGD/PGS, consideringorganization and methodological aspects. Our intention isto provide a detailed methodological view, explaining differ-ent diagnostic strategies based on molecular techniquesthat are currently applied in PGD/PGS from a genomicperspective.
Preconception Genetic Assessment
The referral of couples for PGD/PGS is not a spontaneousdecision. Before PGD/PGS can be performed, there are somelabor-intensive steps. In all cases, the accurate preconceptiongenetic assessment is mandatory in order to clarify familyhistory, compile clinical and genetic reports (if they exist),explain genetic disorders (if it applies), and evaluate thePGD/PGS request and IVF cycle in combination with biopsyprocedures. The genetic risk, the success rates, the risk of mis-diagnosis, and the importance of prenatal diagnosis in case ofpregnancy must be specifically discussed. The complete PGD/PGS process must be explained to the patients and theirqueries must be answered. Finally, alternative reproductiveoptions should be mentioned and a comprehensive informedconsent form must be signed. Usually, these reproductivegenetic studies are being offered to couples by physicians orreproductive medicine professionals from IVF clinics. Inmost cases, genetic studies are performed in collaborationwith an external specialized genetics laboratory. The inter-action of these two groups of experts is essential, not onlyfor the coordination of PGD cycles but also for the exchangeof knowledge in order to offer couples and families the bestdiagnostic options. The need for this interaction has beenpointed out in various reports from the European Union(EU) and the rest of the world [Recommendations of the Euro-pean Societies of Human Genetics and Human Reproductionand Embryology 2006; Soini et al. 2006; Harton et al. 2011a].
In Vitro Fertilization and Embryo Biopsy
A PGD/PGS working scheme includes, in all cases,performing an IVF cycle in order to generate embryos inthe laboratory. Patients undergo a standard cycle of
ovulation induction and each oocyte is microinjected witha single spermatozoon (intracytoplasmic sperm injection,ICSI). Mostly, the embryo biopsy is performed at day +3of in vitro culture, when they are at the 6-8 cell stage, andone or two blastomeres are removed for study. Genetictesting of blastomeres allows extrapolating the embryogenetic status. Afterwards, euploid or free of the diseaseembryos are transferred to the maternal uterus. The timingfor embryo biopsy is under a continuous discussion. Nowa-days, in concordance with the application of microarraytechnology, other times for biopsy have been considered,like polar body [Geraedts et al. 2011] or trophectodermanalysis [Mamas et al. 2012].
Strategies in PGD for Single Gene Disorders
The essential advance in molecular PGD laboratories hasbeen the incorporation of fluorescent polymerase chain reac-tion (f-PCR). Currently, the preferred technique is multiplexf-PCR, nested or heminested [Spits and Sermon 2009].Before describing the different diagnostic approaches, it isimportant to state some particularities of the single cell PCR.
Single cell PCRThe most important limitation in molecular PGD is theamount of DNA in a single cell, of about 6 pg. Performinga nested or heminested PCR allows the reaction to be morespecific. In this case, a first round PCR with outer primers fol-lowed by a second round PCR with fluorescently labelledinner primers allows good results.
There are problems inherent to single cell PCR, like con-tamination. The high number of PCR cycles performed inorder to make the reaction more specific and the limitedamount of DNA, make single cell PCR prone to contami-nation. This can be prevented by physically separating thepre-PCR, PCR, and post-PCR working areas, and havingspecial conditions of sterility [Harton et al. 2011b]. Also,contamination can be from maternal or paternal origin.Removing the cumulus cells around the oocyte or perform-ing ICSI, respectively, avoids this problem. Other sourcesof contamination can be from the PCR or biopsy technician,although it can be detected by making a blank for eachblastomere and using multiplex PCR.
A third problem of single cell PCR is allele drop-out(ADO), the random non-amplification of one of the allelesin a heterozygous sample. It could be a cause of adverse mis-diagnosis [Wilton et al. 2009] as ADO of the mutated allelecould be interpreted as a normal embryo in autosomal domi-nant diseases. Also, in autosomal recessive diseases, ADO ofthe affected allele could alter the number of carrier embryosavailable for transfer, compromising the success of the PGDcycle. There are different hypotheses about the origin ofADO. Nevertheless, there is not a consensus about itsorigin, but the limiting number of DNA copies (two) avail-able in the blastomere appears to be the most accepted cause.Concerning the ways of reducing ADO, increasing the dena-turing temperature and choosing alkaline lysis or proteinase
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K-SDS method can help [Piyamongkol et al. 2003; Thornhillet al. 2005].
Preclinical informativity studies and validationA particularity of PGD for single gene disorders is the needof an informativity study before starting the IVF cycle foreach couple [Alberola et al. 2009, Alberola et al. 2011; Bau-tista-Llácer et al. 2010; Vendrell et al. 2011]. Preclinicalstudies are performed on single cells (lymphocytes, fibro-blasts, or buccal mucosa, depending on the laboratory)from both members of the couple and relatives. Thepurpose of the informativity study is different dependingon the type of diagnostic approach (direct or indirectPGD, see below). Specifically, the informativity test isintended to confirm the disease-causing mutation/s and tofind the informative polymorphic STR markers linked tothe mutation/s or genic regions implicated. STR markersare sequences of two, three, four, or even five nucleotidesrepeated in tandem, typically found in intron regionsthroughout the genome. The number of repeats can varybetween individuals. These STR markers would supportthe diagnosis in case of direct diagnosis PGD. Besides,they are used to establish haplotypes in case of indirectanalysis. Moreover, PCR amplification efficiency and ADOrates on single cells are evaluated as the same optimized con-ditions are expected on blastomeres [Thornhill et al. 2005;Harton et al. 2011b].
These preclinical tests allow establishing the inheritanceof these disorders in each couple and give robustness tothe diagnostic procedure. However, there are couples apply-ing for PGD with no family history, with a de novo disease-causing mutation. In these particular cases, informativitywork-ups based on sperm or oocytes analysis have recentlybeen assessed [Rechitsky et al. 2011].
Indirect PGDThe indirect approach is based on haplotype studies[Renwick 2006; Renwick et al. 2010]. Basically, a set ofSTR markers linked to the disease-causing gene are studiedin the family. STR markers should be, ideally, physicallyclose (less than 1 Mb) to the gene of interest. Also, theymust be at both sides of the gene and be fully informativein order to be able to detect eventual recombination. Theobjective is to identify ‘high risk’ and ‘low risk’ haplotypesin the family, with the purpose of inferring the embryogenetic status (Fig. 1).
This haplotyping strategy allows one to carry out PGDwhen the disease-causing gene is identified but not themutation; when, due to the nature of the mutation, directgenotyping cannot be performed (expansions, duplications,and deletions bigger than 500 base pairs, GC enrichedregions) or even when there exist homologous pseudogenes.Furthermore, in dynamic mutations due to triplet expan-sions, the general strategy consists of amplifying the regionof expansion [Sermon et al. 2001]. Sometimes (e.g., myo-tonic dystrophy) expanded alleles cannot be detected bystandard PCR due to the size of the mutation and only thehealthy allele amplifies. In these cases, the informativity
study prior to PGD is essential in order to determine thehigh and low-risk haplotypes.
One interesting advantage of this methodology is thepossibility of standardization for a particular single genedisorder. A few standardized applications have beenproposed [Gigarel et al. 2008, Fassihi et al. 2010]. Thiswide-scope approach is very useful in cases where DNAfrom the parents and at least one affected member, or suffi-cient unaffected members of a family are available in order toidentify low and high-risk haplotypes.
Indirect PGD is limited in particular cases. Occasionally,there are couples/families with a non-informative STRmarker panel. Moreover, there are genetic regions where itis impossible to find enough STR markers flanking bothsides of the gene of interest. This is an indispensablerequirement in indirect studies in order to detect recombina-tion events and avoid adverse misdiagnosis. Also, familieswhere the affected members are dead, presenting more thanone disease simultaneously, or de novo mutations, require aspecific direct approach. In cases of extremely rare genetic dis-orders with a wide genetic heterogeneity, the setting-up andvalidation of a generic strategy is a laborious task for thevery few cases expected. In our opinion, a case by case strategyis the gold standard option for couples at risk of extremely raredisorders [Vendrell et al. 2011].
Direct PGDThe direct approach allows interrogating the mutation(s)involved in the disease directly on the blastomere. Most ofthe genetic disorders diagnosed by PGD involve a hetero-geneous spectrum of mutations. This has influenced the pro-gress of direct strategies on single cell to fluorescent-based,computer-assisted, and standardized PCR protocols. In thissense, it is possible to establish a general strategy dependingon the mutation type: small deletions, duplications, expan-sions, or insertions (below 500 base pairs) or pointmutations. Informative STR markers flanking the region in-volving the mutation, located at both sides of the gene areincluded in all cases. This confers robustness, as it supportsthe direct genotyping and allows detecting ADO, contami-nation, and eventual recombination.
PGD for small expansions, deletions, duplications, orinsertions is performed by fragment analysis (Fig. 2), gener-ally by means of fluorescent multiplex PCR [Thornhill et al.2005]. In the case of point mutations, minisequencing haspermitted the analysis of specific mutation/s withoutsequencing the entire PCR product [Fiorentino et al.2003]. The region involving the mutation is amplified byPCR and the PCR product is analyzed by minisequencing.Only one nucleotide is elongated, as the minisequencingprimer is designed to terminate one nucleotide before themutation. Therefore, only one dideoxynucleotide of thefour intervening in the reaction is incorporated. Eachdideoxynucleotide is fluorescently labelled with differentfluorochromes emitting in different length waves detectedby capillary electrophoresis (Fig. 3).
There are some other techniques to study point mutationsin PGD, like digestion with restriction endonucleases,
Figure 1. Indirect preimplantation genetic diagnosis (PGD) for spinal muscular atrophy. A) Location and distances to the SMN1 gene of the shorttandem repeat microsatellite markers used. B) Family tree showing the relatives involved in the informativity study and PGD cycle. Both members ofthe couple are carriers of a deletion involving exons 7 and 8 in the SMN1 gene. The dotted line represents the paternal disease-bearing haplotype. Thethick black line illustrates the maternal disease-bearing haplotype. C) Electropherograms of the PGD for spinal muscular atrophy. Examples for twoof the embryos analyzed. The numbers represent fragment sizes in base pairs for every allele. Numbers in bold stand for the alleles associated to thematernal mutation; numbers in bold and italics correspond to the alleles associated to the paternal mutation. Cen: centromere; tel: telomere; Mb:megabases; ADO: allele drop-out.
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Figure 2. Direct preimplantation genetic diagnosis (PGD) for Huntington disease. A) Position and distances to the HTT gene of the short tandemrepeat microsatellite markers involved in the informativity study and PGD cycle. B) Genealogic tree of the family taking part in the study and PGDresults. The arrow designates the consultant affected patient. The thick black line illustrates the expansion-bearing haplotype. C) Electropherogramsof the PGD for Huntington disease. Some examples of the embryos analyzed are displayed. The numbers represent fragment sizes (in base pairs) forevery allele. Numbers corresponding to the disease-bearing allele are in bold. Cen: centromere; tel: telomere; Mb: megabases; ADO: allele drop-out.
Figure 3. Direct PGD for cystic fibrosis. A) Location and distances to the CFTR gene of the short tandem repeat microsatellite markers used in thepreclinical study and preimplantation genetic diagnosis (PGD) cycle. B) Pedigree of the family involved in the study and PGD results. The mothercarries the mutation c.2988 + 1G > A in intron 16 of the CFTR gene. The father carries the mutation c.489 + 1G > T in intron 4 of the CFTR gene. Theaffected daughter carries both mutations in compound heterozygosis. These two positions were minisequenced in the embryos. The dotted line rep-resents the paternal disease-bearing haplotype. The thick black line represents the maternal disease-bearing haplotype. C) Electropherograms of thePGD for cystic fibrosis. Some examples are shown for embryos 1 and 4. The numbers represent fragment sizes for every allele in base pairs. Numbersin bold represent the alleles associated to the maternal mutation; numbers in bold and italics correspond for the alleles associated to the paternalmutation. Cen: centromere; tel: telomere; Mb: megabases; G:guanine; A: adenine; T: timine.
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amplification refractory mutation system (ARMS) or double-ARMS, and real-time PCR. Restriction endonucleases allowdistinguishing between two alleles, normal and mutated,when the mutation is inside the restriction site [Goossenset al. 2000]. The ARMS technique uses one or two highlyspecific primers to detect the mutation of interest by PCR; ifthere is amplification, the mutation is present [Moutou et al.2007]. However, these two techniques are being replaced byminisequencing, as they need very strict controls to be able todistinguish between ADO and non-amplification. Finally,real-time PCR strategy combined with STR-based haplotypeanalysis has been proposed [Traeger-Synodinos 2006]. Briefly,this kind of PCR uses fluorescent probes or dyes and theaccumulation of amplicons produced throughout the progressof PCR reaction is monitored.
Direct approaches in PGD present a series of advantages:i) it allows PGD in couples with de novo mutations or innon-informative couples where standardized methodologyis not possible, ii) it also allows the combination of differenttechniques in recessive diseases and in combined indications(i.e., single gene disorders plus aneuploidy screening bymicroarray technology), and iii) it decreases the possibilityof adverse misdiagnosis in cases where the disease-bearinghaplotypes are wrongly assigned [Altarescu et al. 2008] orwhere a few STR markers are available, or when they arepoorly distributed along the region of study [Wilton et al.2009]. Finally, the application of different direct method-ologies allows increasing the number of diseases for whichPGD is possible. Nevertheless, direct analysis presents animportant challenge, which is to know the mutation respon-sible for a single gene disorder. This is complicated in dis-orders of very low prevalence or in polygenic diseases.
Whole Genome Amplification
The PCR-based methods presented have many advantages,but there are still some limitations. These methods cannotmeet the needs of whole genome research. It is well-knownthat the limiting factor of any technique applied to PGD isthe amount of DNA available. One approach that increasesthe quantity of DNA from one single cell is whole genomeamplification (WGA) technologies [Coskun and Alsmadi2007]. Basically, it is based on the general amplification ofthe whole genomic sequence, yielding microgram quantitiesof DNA while respecting the original sequence. There arePCR and non-PCR methods of WGA, thoroughly reviewedby Zheng and coworkers [2011]. WGA allows combiningdifferent indications in the same PGD cycle, like HLA hap-lotyping or microarray technology like comparative genomichybridization (CGH) plus detection of a single gene disorder[Handyside et al. 2004; Brezina 2011].
HLA-Compatible Embryos
The PGD strategy used by couples with a son requiringhematopoietic transplantation from a HLA compatibledonor is particularly remarkable. Moreover, if the disorderis genetic and parents are carriers of the mutation-causing
disease, PGD is more complex. In these cases, PGD for themonogenic disorder plus HLA haplotyping is performed,in order to select healthy and compatible embryos with theprevious affected sibling [Fiorentino et al. 2004; Fiorentinoet al. 2005; Rechitsky 2006; Van de Velde et al. 2009). Aninformativity study is required in order to identify the infor-mative STR markers. Eight to 13 informative STRs areselected (according to different groups), distributed alongthe HLA region, spanning 40 Mb on chromosome 6p. Mul-tiple displacement amplification (MDA) or multiplex PCR(depending on the group) follows, and the work-up is vali-dated on single cells isolated from the couple, togetherwith mutation detection of the disease plus STRs supportingthe diagnosis or haplotypes (Fig. 4).
Molecular Strategies for Chromosomal Studies
Nowadays, molecular biology approaches are systematicallyreplacing FISH techniques for some PGD/PGS indications.There has been a great debate about the usefulness of PGS,where up to 12 chromosomes are tested. This debate is stillopen. However, the main conclusion is that FISH-basedPGS does not increase the pregnancy rate for advancedmaternal age, as there might be other chromosomes involvedcausing aneuploidy that are not checked [Harper et al.2010a]. CGH has been proposed as an alternative to FISH,where all chromosomes are analyzed by using metaphasechromosomes as the template [Wilton 2005; Obradorset al. 2008]. However, it is time-consuming, labor intensive,and requires the appropriate skills and expertise in order tointerpret the results. Recently, two types of microarray-basedtechniques are being clinically used in PGS and PGD forchromosomal rearrangements: i) array-CGH (aCGH)[Gutiérrez-Mateo et al. 2011] and ii) single-nucleotidepolymorphism arrays (aSNP) [Johnson et al. 2010; Treffet al. 2010].
In aCGH, whole DNA pre-amplified from single blasto-mere (or trophectodermic cells) and normal referencesamples are labelled using different fluorophores (red andgreen) and competitively hybridized to bacterial artificialchromosomes (BAC) clones printed on a glass slide.Each BAC clone corresponds to a specific segment of achromosome. The ratio of the fluorescence intensity is calcu-lated in order to find copy number variations. Computersoftware analyzes the ratio of red/green fluorescence oneach clone calculating loss or gain of the particular regionas the log2 ratio. Technical improvements have allowedreducing the time, and a diagnosis is given in approximately24 hours. The software shows the information correspond-ing to the signal of the sample across the whole chromosome(Fig. 5). This application yields a wide-scope information atchromosome level, detecting numerical and structuralchromosome abnormalities [Fiorentino et al. 2011], evensmall losses in chromosome arms. The limitation of aCGHis that polyploidy, haploidy, and balanced structuralrearrangements cannot be detected. Simultaneously, aPCR-based strategy has been developed to detect reciprocaland Robertsonian translocation carriers in PGD [Fiorentino
et al. 2010; Traversa et al. 2010]. Nevertheless, normal, ba-lanced, and unbalanced translocations carrier embryos canbe distinguished with a novel molecular approach[Shamash et al. 2011] by using preimplantation genetic hap-lotyping [Renwick et al. 2010].
aSNP platforms let us obtain information concerninghow much of each chromosome was inherited. SNPs arechanges in only one nucleotide in the DNA sequencewithin the population. These changes permit one to differen-tiate one chromosome from another in any person. In this
sense, aSNP have been introduced for aneuploidy detectionin the context of PGS, giving extra information in relationto parent-of-origin and, in some cases, it offers informationrelated to uniparental disomy. However, the major limitationof this approach is that data analysis is labour-intensive andrequires optimized protocols. In this sense, a very interestingdebate comparing BACs versus SNPs-based arrays has beendiscussed recently [Bisignano et al. 2011; Handyside 2011].Unfortunately, publications of clinical applications in thefield of PGS are still scarce.
Figure 4. Combined preimplantation genetic diagnosis (PGD) for Fanconi anemia combined with human leukocyte antigen (HLA) matching. A)Location of the short tandem repeat microsatellite marker D16S520 with respect to the FANCA gene on chromosome 16. B) Genealogic tree of thefamily involved in the preclinical study and PGD results. The arrow indicates the consultant patient. Haplotypes for the HLA region on chromosome6 plus Fanconi anemia mutations positions on chromosome 16 were established. The discontinuous line and the fine line represent the HLA hap-lotypes from themother and the father, respectively, that embryos should inherit in order to be HLA compatible with the affected son. The thick blackline symbolizes the disease-bearing haplotype for the paternal mutation (c.3788_3790delTCT) and the dotted line denotes the disease-bearing hap-lotype for the maternal mutation (c.4130C > G). Mutation c.3788_3790delTCT was determined by fragment analysis and position c.4130 of theFANCA gene was minisequenced. The genetic status for every embryo obtained is differentiated between HLA compatibility and Fanconi anemiadiagnosis. Cen: centromere; tel: telomere; Mb: megabases; C: cytosine; G:guanine.
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What’s Next?
The diagnostic strategies in PGS/PGD have experienced arapid evolution. Nowadays, the different strategies representa consolidated approach for single (or a few) cell geneticanalysis. As it has occurred in other genetic applications,three basic points should be considered in the near future:i) standardization of the analytical methods, ii) analysis ofcost-effectiveness, and iii) application of emerging techno-logical advances. Related to standardization, the application
of international quality standards to PGD laboratories, asthe ISO 9001:2008 certification [Vendrell et al. 2009] andthe ISO 15189 [Harper et al. 2010c] or equivalent accredita-tion, are extremely important in an under-regulated diag-nostic approach like PGD/PGS. In this sense, organizationis essential. Aspects as licensing, professional qualificationof personnel, and inclusion/exclusion criteria for patientreferrals or genetic conditions that can be diagnosed withPGD have recently been thoroughly revised [Harton et al.2011a]. From the regulatory point of view, every country
Figure 5. Example of blastomere array- comparative genomic hybridization charts. Chromosomal position is represented on the X-axis and theLog2 ratio Ch1/Ch2 (the ratio of red/green fluorescence intensity on each clone) on the Y-axis. A) Female blastomere with gain of chromosome3(p14.2→qter) and loss of chromosome 11(q12.3→qter). The father carries a balanced translocation with chromosome formula: 46,XY;t(3;11)(p21;q13). B) Array profile showing a female cell with gain of chromosome 4. C) Normal female profile.
regulates these practices in a more or less specific way and insome cases, there are no explicit laws. In addition, majordifferences have generated a significant flow of patientsbetween different countries. In this scenario, it is verycomplex to determine what actions must be taken toensure quality standards in PGD/PGS. Various scientificsocieties [The Practice Committee of American Society forReproductive Medicine 2004; The PreimplantationGenetic Diagnosis International Society (PGDIS) 2004;2008; Thornhill et al. 2005; Harton et al. 2011a;Harton et al. 2011b; Harton et al. 2011c; Harton et al.2011d] have proposed different guidelines in order tostandardize and achieve the highest levels of optimization,reproducibility, and control of risks in genetic diagnosison single cells.
Accreditation by national bodies is the formal recognitionof technical competence and the compliance of the inter-national quality standards. Concretely, the norm ISO15189 treats two main aspects concerning medical labora-tories: i) management and ii) technical requirements. Theglobal process is externally audited by recognized experts,considering pre-analytical, analytical, and post-analyticalphases, in the context of a quality management system andcombined with the participation in inter-laboratory ringtests. The aim is to implement and analyze the ‘quality indi-cators’, systematically monitored and documented. The finalobjective is to continue improving.
The cost-effectiveness of these analytical processes is notirrelevant. PGD/PGS is a part of a complex and costlyprocess, with highly trained professionals (clinicians, embry-ologists, and geneticists experts), and with a high technologi-cal component. Globally, there are several strategies relatedto funding the IVF-PGD/PGS treatment: national publicprograms, private clinics, private/public copayment, andinsurance coverage. Different countries maintain medical,ethical, and legal discussions concerning IVF-PGD/PGStreatments. However, there are few studies about the econ-omic impact of PGD applications on the healthcaresystems. Tur-Kaspa and co-workers [2010] presented avery interesting study in this sense, taking cystic fibrosis asan example. They conclude that “a national IVF-PGDprogram is a highly cost effective novel strategy of modernpreventive medicine.” In agreement with the authors, IVF-PGD could play a significant role reducing incidence ofinherited disorders, and perhaps, it should be a principalissue in the debate concerning early diagnosis and preventivemedicine.
Regarding emerging techniques, the next step presentsnew challenges. Working with single cells is difficult dueto the limiting quantity of DNA. WGA technologies havepermitted applying haplotyping to aSNP for PGD [Handy-side et al. 2010; Handyside 2011]. This new ‘karyomap’identifies the parental origin of each chromosome andpermits analysis of recombination patterns. Besides, thistechnique offers the possibility of detecting numerical andstructural chromosomal imbalances. This new SNP-basedkaryomapping combined with WGA in PGD is at theinitial stage and forthcoming validation in single cell is
necessary in order to be established as a new tool indiagnosis.
Combining indications as single-gene disorders detectionplus aneuploidy screening has been suggested. Recently, thisnew PGD indication has been proposed by trophectodermbiopsy [Brezina et al. 2011]. This approach introduces adouble novelty: the timing of biopsy (on day +5) and thecombined genetic study. In this direction, our group pre-sented, very recently (in the past meeting of The SpanishAssociation for the Study of Reproductive Biology,ASEBIR), the use of WGA methods including specificprimers to amplify the genetic regions of interest, permittingthe simultaneous application of aCGH and PCR-based tech-nologies in one single blastomere.
Furthermore, the application of genomic strategies basedon high throughput sequencing platforms is a very promis-ing approach [Mardis 2011]. Nowadays, Next GenerationSequencing (NGS) is revolutionizing the classical conceptof genetic diagnosis, offering a ‘genomic view’ in the com-prehension of gene expression profiles, DNA methylationchanges, and identification of non-coding RNA expressionprofiles. From the ‘embryo-genomics’ point of view, thewhole-transcriptome analysis on single mouse blastomere[Tang et al. 2009], and the comprehension of methylome(methylation patterns) based on high throughput sequen-cing from preimplantation embryos [Ma et al. 2012] rep-resent the first step in this new genomic era.
Final Comments
Historically, PGD/PGS have been closely linked to the devel-opment of assisted reproduction technologies. In fact, thefirst publications appeared in the context of IVF units. Cur-rently, most IVF clinics offer this highly specialized geneticstudy in association with a team of geneticists. This success-ful union will continue in order to achieve new milestones.In fact, from the assisted reproduction point of view, theemergence of new methods of cryopreservation (vitrifica-tion), new embryo culture conditions, different timing ofbiopsy (polar body or trophectoderm), and embryo transferstrategies, could change the classical schedules of PGD/PGS.In conclusion, rapid advances in genetics and genomics arechanging genetic testing and screening in preconception,prenatal, and newborn care. It is fundamentally importantthat assisted reproduction clinicians and experts who arerecommending such studies have a deep knowledge of thecurrent sensitivity and specificity of tests, thus they couldperform a correct referral for complex results and precon-ception genetic assessment.
Author Contributions: The authors contributed equally indesigning and performing the experiments, analysis of dataand writing of the manuscript.
Declaration of interest: The authors declare the absence ofany financial or commercial interest in reference to the sub-mitted material. The authors would not economically benefitfrom publication of the manuscript. All data are presented
298 X. Vendrell and R. Bautista-Llácer
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Notice of CorrectionThe version of this article published online ahead of print on 3 AUG 2012 contained a number of minortypographical errors. These have been corrected for this version
