Voltar
Revisão Acesso aberto Revisado por pares
BIBTEX RIS

Molecular Diagnostics in Preimplantation Genetic Diagnosis

2002; Elsevier BV; Volume: 4; Issue: 1 Linguagem: Inglês

10.1016/s1525-1578(10)60676-9

ISSN

1943-7811

Autores

Alan R. Thornhill, Karen Snow,

Tópico(s)

Parvovirus B19 Infection Studies

Resumo

Preimplantation genetic diagnosis (PGD) is a procedure that allows embryos to be tested for genetic disorders before they enter the uterus and before pregnancy has begun. Embryos obtained by in vitro fertilization undergo a biopsy procedure in which one or two cells are removed and tested for a specific disorder. If the cell is unaffected, the embryo from which it was taken is judged to be free of the disorder. The embryo can then be transferred to the uterus to initiate pregnancy. Couples whose children are at increased risk for a specific genetic disorder can benefit from PGD. Some of these couples may have affected family members or family ancestry that puts them at high risk for transmitting a particular disorder to their offspring. PGD is an alternative to prenatal tests such as amniocentesis or chorionic villus sampling and since it is performed before a pregnancy has begun, it may be more acceptable to couples who have either had an affected child, previous termination of pregnancy, or who have objections to termination of pregnancy. PGD tests have largely focused on two methodologies: fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR). This review will focus on the use of PCR-based methodologies to diagnose single gene disorders in single cells; specifically describing the characteristics and limitations of single cell PCR and mutation detection strategies which have been developed for use in clinical PGD. The hundreds of cycles of preimplantation diagnosis performed to date have resulted in the birth of several hundred healthy children.1ESHRE preimplantation genetic diagnosis (PGD) consortium: data collection II.Hum Reprod. 2000; 15: 2673-2683Crossref PubMed Google Scholar As shown in Table 1, the genetic conditions for which PGD has been applied are numerous and the various methods used for diagnosis reflect the heterogeneity of causative mutations.Table 1Strategies for PCR-Based Tests Used for Clinical Preimplantation Genetic DiagnosisMethodDisorder to be diagnosedMutation typeSingle PCR, agarose gel (+/− Y band)X-linked disorders2Handyside AH Kontogianni EH Hardy K Winston RM Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification.Nature. 1990; 344: 768-770Crossref PubMed Scopus (658) Google ScholarVarious (gender determination to exclude hemizygotes)Nested PCR, agarose gel (+/− X/Y)X-linked disorders6Levinson G Keyvanfar K Wu JC Fugger EF Fields RA Harton GL Palmer FT Sisson ME Starr KM Dennison-Lagos L Calvo L Sherins RJ Bick D Schulman JD Black SH DNA-based X-enriched sperm separation as an adjunct to preimplantation genetic testing for the prevention of X-linked disease.Hum Reprod. 1995; 10: 979-982PubMed Google ScholarVarious (gender determination to exclude hemizygotes)Nested PCR, heteroduplexingCystic fibrosis22Ray PF Ao A Taylor DM Winston RML Handyside AH Assessment of the reliability of single blastomere analysis for preimplantation diagnosis of the ΔF508 deletion causing cystic fibrosis in clinical practice.Prenat Diagn. 1998; 18: 1402-1412Crossref PubMed Scopus (45) Google Scholar38Liu J Lissens W Silber SJ Devroey P Liebaers I Van Steirteghem A Birth after preimplantation diagnosis of the cystic fibrosis delta F508 mutation by polymerase chain reaction in human embryos resulting from intracytoplasmic sperm injection with epididymal sperm.JAMA. 1994; 272: 1858-1860Crossref PubMed Google Scholar44Ao A Ray P Harper J Lesko J Paraschos T Atkinson G Soussis I Taylor D Handyside A Hughes M Winston RM Clinical experience with preimplantation genetic diagnosis of cystic fibrosis (delta F508).Prenat Diagn. 1996; 16: 137-142Crossref PubMed Scopus (47) Google Scholar75Handyside AH Lesko JG Tarin JJ Winston RM Hughes MR Birth of a normal girl after in vitro fertilization and preimplantation diagnostic testing for cystic fibrosis.N Engl J Med. 1992; 327: 905-909Crossref PubMed Google Scholar Tay-Sachs disease111Gibbons WE Gitlin SA Lanzendorf SE Kaufmann RA Slotnick RN Hodgen GD Preimplantation genetic diagnosis for Tay-Sachs disease: successful pregnancy after pre-embryo biopsy and gene amplification by polymerase chain reaction.Fertil Steril. 1995; 63: 723-728Abstract Full Text PDF PubMed Google Scholar3 bp deletion (ΔF508) 4 bp insertionNested PCR, allele-specific amplificationRhD blood typing3Avner R Reubinoff BE Simon A Zentner BS Friedmann A Mitrani Rosenbaum S Laufer N Management of rhesus isoimmunization by preimplantation genetic diagnosis.Mol Hum Reprod. 1996; 2: 60-62Crossref PubMed Google Scholar Myotonic dystrophy127Sermon K Lissens W Joris H Seneca S Desmyttere S Devroey P Van Steirteghem A Liebaers I Clinical application of preimplantation diagnosis for myotonic dystrophy.Prenat Diagn. 1997; 17: 925-932Crossref PubMed Scopus (48) Google Scholar+/− RhD gene determines Rh status Expansion of (CTG)n trinucleotide repeatNested PCR, restriction enzymeCystic fibrosis,23Avner R Laufer N Safran A Kerem BS Friedmann A Mitrani Rosenbaum S Preimplantation diagnosis of cystic fibrosis by simultaneous detection of the W1282X and ΔF508 mutations.Hum Reprod. 1994; 9: 1676-1680PubMed Google Scholar Beta thalassemia,83Kuliev A Rechitsky S Verlinsky O Ivakhnenko V Evsikov S Wolf G Angastiniotis M Georghiou D Kukharenko V Strom C Verlinsky Y Preimplantation diagnosis of thalassemias.J Assist Reprod Genet. 1998; 15: 219-225Crossref PubMed Scopus (79) Google Scholar Marfan syndrome,107Blaszczyk A Tang YX Dietz HC Adler A Berkeley AS Krey LC Grifo JA Preimplantation genetic diagnosis of Marfan's syndrome.J Assist Reprod Genet. 1998; 15: 281-284Crossref PubMed Scopus (28) Google Scholar Epidermolysis Bullosa,100Cserhalmi-Friedman PB Tang Y Adler A Krey L Grifo JA Christiano AM Preimplantation genetic diagnosis in two families at risk for recurrence of Herlitz junctional epidermolysis bullosa.Exp Dermatol. 2000; 9: 290-297Crossref PubMed Scopus (32) Google Scholar Lesch-Nyhan syndrome,101Ray PF Harper JC Ao A Taylor DM Winston RM Hughes M Handyside AH Successful preimplantation genetic diagnosis for sex -linked Lesch-Nyhan syndrome using specific diagnosis.Prenat Diagn. 1999; 19: 1237-1241Crossref PubMed Scopus (22) Google Scholar Sickle cell anemia,102Xu K, Shi ZM, Veeck LL, Hughes MR, Rosenwaks Z: First unaffected pregnancy using preimplantation genetic diagnosis for sickle cell anemia. JAMA 281:1701–1706Google Scholar Fanconi's anemia,103Verlinsky Y Rechitsky S Schoolcraft W Strom C Kuliev A Preimplantation diagnosis for Fanconi anemia combined with HLA matching.JAMA. 2001; 285: 3130-3133Crossref PubMed Google Scholar Ornithine transcarbamylase deficiency,104Ray PF Gigarel N Bonnefont JP Attie T Hamamah S Frydman N Vekemans M Frydman R Munnich A First specific preimplantation genetic diagnosis for ornithine transcarbamylase deficiency.Prenat Diagn. 2000; 20: 1048-1054Crossref PubMed Scopus (28) Google Scholar Spinal muscular atrophy108Dreesen JC Bras M de Die-Smulders C Dumoulin JC Cobben JM Evers JL Smeets HJ Geraedts JP Preimplantation genetic diagnosis of spinal muscular atrophy.Mol Hum Reprod. 1998; 4: 881-885Crossref PubMed Scopus (43) Google Scholar109Fallon L Harton GL Sisson ME Rodriguez E Field LK Fugger EF Geltinger M Sun Y Dorfmann A Schoener C Bick D Schulman J Levinson G Black SH Preimplantation genetic diagnosis for spinal muscular atrophy type I.Neurology. 1999; 53: 1087-1090Crossref PubMed Google Scholar110Daniels G Pettigrew R Thornhill A Abbs S Lashwood A O'Mahoney F Mathew C Handyside A Braude P Six unaffected livebirths following preimplantation diagnosis for spinal muscular atrophy.Mol Hum Reprod. 2001; 7: 995-1000Crossref PubMed Google ScholarVarious point mutations Deletion. Distinguish between gene and pseudogeneNested PCR, restriction enzyme (2 mutations in 1 fragment)Skin fragility ectodermal dysplasia syndrome66Thornhill AR Pickering SJ Whittock N Caller J Andritsos V Handyside AH Braude PR Eady RA McGrath J Preimplantation genetic diagnosis of compound heterozygous mutations leading to ablation of Plakophilin-1 (PKP1) and resulting in skin fragility ectodermal dysplasia syndrome.Prenat Diagn. 2000; 20: 1055-1062Crossref PubMed Scopus (18) Google ScholarAllows detection of ADOWhole genome amplification and comparative genome hybridizationAneuploidy screening96Wilton L Williamson R McBain J Edgar D Voullaire L Birth of a healthy infant after preimplantation confirmation of euploidy by comparative genomic hybridization.N Engl J Med. 2001; 21: 1537-1541Crossref Scopus (117) Google ScholarNAWhole genome amplification (PEP)Familial adenomatous polyposis coli60Ao A Wells D Handyside AH Winston RM Delhanty JD Preimplantation genetic diagnosis of inherited cancer: familial adenomatous polyposis coli.J Assist Reprod Genet. 1998; 15: 140-144Crossref PubMed Scopus (86) Google ScholarMultiple analyses from each sampleNested PCR, linked markersDuchenne muscular dystrophy10Liu J Lissens W Van Broeckhoven C Lofgren A Camus M Liebaers I Van Steirteghem A Normal pregnancy after preimplantation DNA diagnosis of a dystrophin gene deletion.Prenat Diagn. 1995; 15: 351-358Crossref PubMed Google Scholar125Lee SH Kwak IP Cha KE Park SE Kim NK Cha KY Preimplantation diagnosis of non-deletion Duchenne muscular dystrophy (DMD) by linkage polymerase chain reaction analysis.Mol Hum Reprod. 1988; 4: 345-349Crossref Scopus (24) Google Scholar Ornithine transcarbamylase deficiency104Ray PF Gigarel N Bonnefont JP Attie T Hamamah S Frydman N Vekemans M Frydman R Munnich A First specific preimplantation genetic diagnosis for ornithine transcarbamylase deficiency.Prenat Diagn. 2000; 20: 1048-1054Crossref PubMed Scopus (28) Google ScholarExon deletions Point mutation (linked marker for ADO detection)Nested PCR, SSCPFamilial Adenomatous Polyposis Coli60Ao A Wells D Handyside AH Winston RM Delhanty JD Preimplantation genetic diagnosis of inherited cancer: familial adenomatous polyposis coli.J Assist Reprod Genet. 1998; 15: 140-144Crossref PubMed Scopus (86) Google ScholarPoint mutationNested PCR, direct cycle sequencingSkin fragility ectodermal dysplasia syndrome66Thornhill AR Pickering SJ Whittock N Caller J Andritsos V Handyside AH Braude PR Eady RA McGrath J Preimplantation genetic diagnosis of compound heterozygous mutations leading to ablation of Plakophilin-1 (PKP1) and resulting in skin fragility ectodermal dysplasia syndrome.Prenat Diagn. 2000; 20: 1055-1062Crossref PubMed Scopus (18) Google ScholarPoint mutations (cycle sequencing to confirm restriction digest)Nested PCR, DGGEBeta thalassemia115Kanavakis E Vrettou C Palmer G Tzetis M Mastrominas M Traeger-Synodinos J Preimplantation genetic diagnosis in 10 couples at risk for transmitting beta-thalassaemia major: clinical experience including the initiation of six singleton pregnancies.Prenat Diagn. 1999; 19: 1217-1222Crossref PubMed Scopus (44) Google ScholarPoint mutationsHeminested PCR, site specific mutagenesisRetinitis pigmentosa99Strom CM Rechitsky S Wolf G Cieslak J Kuliev A Verlinsky Y Preimplantation diagnosis of autosomal dominant retinitis pigmentosum using two simultaneous single cell assays for a point mutation in the rhodopsin gene.Mol Hum Reprod. 1998; 4: 351-355Crossref PubMed Scopus (10) Google Scholar Ornithine transcarbamylase deficiency104Ray PF Gigarel N Bonnefont JP Attie T Hamamah S Frydman N Vekemans M Frydman R Munnich A First specific preimplantation genetic diagnosis for ornithine transcarbamylase deficiency.Prenat Diagn. 2000; 20: 1048-1054Crossref PubMed Scopus (28) Google ScholarPoint mutation Point mutationHeminested PCR, allele dependent length polymorphismRetinitis pigmentosa99Strom CM Rechitsky S Wolf G Cieslak J Kuliev A Verlinsky Y Preimplantation diagnosis of autosomal dominant retinitis pigmentosum using two simultaneous single cell assays for a point mutation in the rhodopsin gene.Mol Hum Reprod. 1998; 4: 351-355Crossref PubMed Scopus (10) Google ScholarPoint mutationNested multiplex PCR (including linked markers)Marfan syndrome119Harton GL Tsipouras P Sisson ME Starr KM Mahoney BS Fugger EF Schulman JD Kilpatrick MW Levinson G Black SH Preimplantation genetic testing for Marfan syndrome.Mol Hum Reprod. 1996; 2: 713-715Crossref PubMed Google Scholar Epidermolysis Bullosa100Cserhalmi-Friedman PB Tang Y Adler A Krey L Grifo JA Christiano AM Preimplantation genetic diagnosis in two families at risk for recurrence of Herlitz junctional epidermolysis bullosa.Exp Dermatol. 2000; 9: 290-297Crossref PubMed Scopus (32) Google Scholar Beta thalassemia83Kuliev A Rechitsky S Verlinsky O Ivakhnenko V Evsikov S Wolf G Angastiniotis M Georghiou D Kukharenko V Strom C Verlinsky Y Preimplantation diagnosis of thalassemias.J Assist Reprod Genet. 1998; 15: 219-225Crossref PubMed Scopus (79) Google ScholarUnknown mutation Monitor allele dropoutNested multiplex PCR (including linked and non-linked markers)Sickle cell anemia,41Rechitsky S Strom C Verlinsky O Amet T Ivakhnenko V Kukharenko V Kuliev A Verlinsky Y Allele dropout in polar bodies and blastomeres.J Assist Reprod Genet. 1998; 15: 253-257Crossref PubMed Scopus (85) Google Scholar hemophilia B,41Rechitsky S Strom C Verlinsky O Amet T Ivakhnenko V Kukharenko V Kuliev A Verlinsky Y Allele dropout in polar bodies and blastomeres.J Assist Reprod Genet. 1998; 15: 253-257Crossref PubMed Scopus (85) Google Scholar cystic fibrosis,41Rechitsky S Strom C Verlinsky O Amet T Ivakhnenko V Kukharenko V Kuliev A Verlinsky Y Allele dropout in polar bodies and blastomeres.J Assist Reprod Genet. 1998; 15: 253-257Crossref PubMed Scopus (85) Google Scholar Gaucher disease,61Rechitsky S Strom C Verlinsky O Amet T Ivakhnenko V Kukharenko V Kuliev A Verlinsky Y Accuracy of preimplantation diagnosis of single-gene disorders by polar body analysis of oocytes.J Assist Reprod Genet. 1999; 16: 192-198Crossref PubMed Scopus (51) Google Scholar Long chain 3-hydroxyacyl-CoA dehydrogenase deficiency61Rechitsky S Strom C Verlinsky O Amet T Ivakhnenko V Kukharenko V Kuliev A Verlinsky Y Accuracy of preimplantation diagnosis of single-gene disorders by polar body analysis of oocytes.J Assist Reprod Genet. 1999; 16: 192-198Crossref PubMed Scopus (51) Google ScholarMonitor allele dropout and contaminationFluorescent PCR, allele size (fragment analysis)Huntington disease32Sermon K Goossens V Seneca S Lissens W De Vos A Vandervorst M Van Steirteghem A Liebaers I Preimplantation diagnosis for Huntington's disease (HD): clinical application and analysis of the HD expansion in affected embryos.Prenat Diagn. 1998; 18: 1427-1436Crossref PubMed Scopus (94) Google Scholar Cystic fibrosis106Goossens V Sermon K Lissens W Vandervorst M Vanderfaeillie A De Rijcke M De Vos A Henderix P Van De Velde H Van Steirteghem A Liebaers I Clinical application of preimplantation genetic diagnosis for cystic fibrosis.Prenat Diagn. 2000; 20: 571-581Crossref PubMed Scopus (32) Google Scholar Myotonic dystrophy55Sermon K De Vos A Van de Velde H Seneca S Lissens W Joris H Vandervorst M Van Steirteghem A Liebaers I Fluorescent PCR and automated fragment analysis for the clinical application of preimplantation genetic diagnosis of myotonic dystrophy (Steinert's disease).Mol Hum Reprod. 1998; 4: 791-796Crossref PubMed Google Scholar Fragile X syndrome128Sermon K Seneca S Vanderfaeillie A Lissens W Joris H Vandervorst M Van Steirteghem A Liebaers I Preimplantation diagnosis for fragile X syndrome based on the detection of the non-expanded paternal and maternal CGG.Prenat Diagn. 1999; 19: 1223-1230Crossref PubMed Scopus (50) Google ScholarExpansion of (CAG)n trinucleotide repeat 3 bp deletion Expansion of (CTG)n trinucleotide repeat Expansion of (CGG)n trinucleotide repeatFluorescent PCR, SSCPMedium chain acyl CoA dehydrogenase deficiency76Ioulianos A Wells D Harper JC Delhanty JD A successful strategy for preimplantation diagnosis of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency.Prenat Diagn. 2000; 20: 593-598Crossref PubMed Scopus (17) Google ScholarPoint mutationFluorescent PCR, ARMSSpinal muscular atrophy77Moutou C Gardes N Rongieres C Ohl J Bettahar-Lebugle K Wittemer C Gerlinger P Viville S Allele-specific amplification for preimplantation genetic diagnosis (PGD) of spinal muscular atrophy.Prenat Diagn. 2001; 21: 498-503Crossref PubMed Scopus (30) Google ScholarExon deletion in gene but not pseudogeneFluorescent PCR, restriction analysisCongenital adrenal hyperplasia,78Van de Velde H Sermon K De Vos A Lissens W Joris H Vandervorst M Van Steirteghem A Liebaers I Fluorescent PCR and automated fragment analysis in preimplantation genetic diagnosis for 21-hydroxylase deficiency in congenital adrenal hyperplasia.Mol Hum Reprod. 1999; 5: 691-696Crossref PubMed Scopus (22) Google Scholar osteogenesis imperfecta,105De Vos A Sermon K Van de Velde H Joris H Vandervorst M Lissens W De Paepe A Liebaers I Van Steirteghem A Two pregnancies after preimplantation genetic diagnosis for osteogenesis imperfecta type I and type IV.Hum Genet. 2000; 106: 605-613Crossref PubMed Google Scholar medium chain acyl CoA dehydrogenase deficiency,33Sermon K Henderix P Lissens W De Vos A Vandervorst M Vanderfaeillie A Vamos E Van Steirteghem A Liebaers I Preimplantation genetic diagnosis for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency.Mol Hum Reprod. 2000; 6: 1165-1168Crossref PubMed Google Scholar Sickle cell anemia34De Rycke M Van de Velde H Sermon K Lissens W De Vos A Vandervorst M Vanderfaeillie A Van Steirteghem A Liebaers I Preimplantation genetic diagnosis for sickle-cell anemia and for beta-thalassemia.Prenat Diagn. 2001; 21: 214-222Crossref PubMed Scopus (43) Google ScholarPoint mutationsFluorescent PCR, restriction analysis (2 mutations in 1 fragment)Beta thalassemia34De Rycke M Van de Velde H Sermon K Lissens W De Vos A Vandervorst M Vanderfaeillie A Van Steirteghem A Liebaers I Preimplantation genetic diagnosis for sickle-cell anemia and for beta-thalassemia.Prenat Diagn. 2001; 21: 214-222Crossref PubMed Scopus (43) Google ScholarPoint mutations, small deletionMultiplex Fluorescent PCRBeta thalassemia34De Rycke M Van de Velde H Sermon K Lissens W De Vos A Vandervorst M Vanderfaeillie A Van Steirteghem A Liebaers I Preimplantation genetic diagnosis for sickle-cell anemia and for beta-thalassemia.Prenat Diagn. 2001; 21: 214-222Crossref PubMed Scopus (43) Google ScholarPoint mutations, small deletionMultiplex Fluorescent PCR (including unlinked marker)Myotonic dystrophy130Piyamongkol W Harper JC Sherlock JK Doshi A Serhal PF Delhanty JD Wells D A successful strategy for preimplantation genetic diagnosis of myotonic dystrophy using multiplex fluorescent PCR.Prenat Diagn. 2001; 21: 223-232Crossref PubMed Scopus (44) Google ScholarExpansion of (CTG)n trinucleotide repeat/contamination controlMultiplex Fluorescent PCR (including linked marker)Medium chain acyl CoA dehydrogenase deficiency76Ioulianos A Wells D Harper JC Delhanty JD A successful strategy for preimplantation diagnosis of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency.Prenat Diagn. 2000; 20: 593-598Crossref PubMed Scopus (17) Google ScholarMaternal mutation unknownFluorescent PCR, linked markers onlyFragile X syndrome120Apessos A Abou-Sleiman PM Harper JC Delhanty JD Preimplantation genetic diagnosis of the fragile X syndrome by use of linked polymorphic markers.Prenat Diagn. 2001; 21: 504-511Crossref PubMed Scopus (33) Google Scholar Marfan syndrome121Sermon K Lissens W Messiaen L Bonduelle M Vandervorst M Van Steirteghem A Liebaers I Preimplantation genetic diagnosis of Marfan syndrome with the use of fluorescent polymerase chain reaction and the automated laser fluorescence DNA sequencer.Fertil Steril. 1999; 71: 163-166Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar Charcot Marie Tooth disease45De Vos A Sermon K Van de Velde H Joris H Vandervorst M Lissens W Mortier G DeSutter P Lofgren A Van Broeckhoven C Liebaers I Van Steirteghem A Pregnancy after preimplantation genetic diagnosis for Charcot-Marie-Tooth disease type 1A.Mol Hum Reprod. 1998; 4: 978-984Crossref PubMed Scopus (32) Google Scholar Cystic fibrosis123Eftedal I Schwartz M Bendtsen H Andersen AN Ziebe S Single intragenic microsatellite preimplantation genetic diagnosis for cystic fibrosis provides positive allele identification of all CFTR genotypes for informative couples.Mol Hum Reprod. 2001; 7: 307-312Crossref PubMed Google ScholarExpanded (CGG)n repeat (refractory to PCR) Unknown mutation Gene duplication Heterogeneous mutations Open table in a new tab The first clinical application of PGD used a generic PCR protocol for gender determination to avoid the transfer of male embryos which have a 50% probability of being affected by an X-linked recessive disorder. Gender was determined in a single blastomere by a single round of PCR using primers for Y-chromosome specific repetitive DNA sequences. The presence of Y-specific PCR amplification products was indicative of a male embryo and the absence of products was scored as female.2Handyside AH Kontogianni EH Hardy K Winston RM Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification.Nature. 1990; 344: 768-770Crossref PubMed Scopus (658) Google Scholar Although this approach had some success, a misdiagnosis, presumably due to amplification failure, did occur and emphasized the challenges inherent in single cell analysis and, more specifically, the danger in relying on the absence of amplification to diagnose genotype.3Avner R Reubinoff BE Simon A Zentner BS Friedmann A Mitrani Rosenbaum S Laufer N Management of rhesus isoimmunization by preimplantation genetic diagnosis.Mol Hum Reprod. 1996; 2: 60-62Crossref PubMed Google Scholar Subsequently, PCR protocols for preimplantation gender determination were refined to include primer sets which simultaneously amplify sequences common to both sex chromosomes (for example single copy genes such as ZFX/ZFY,4Chong SS Kristjansson K Cota J Handyside AH Hughes MR Preimplantation prevention of X-linked disease: reliable and rapid sex determination of single human cells by restriction analysis of simultaneously amplified ZFX and ZFY sequences.Hum Mol Genet. 1993; 2: 1187-1191Crossref PubMed Google ScholarAMELX/AMELY,5Nakahori Y Hamano K Iwaya M Nakagome Y Sex identification by polymerase chain reaction using X-Y homologous primer.Am J Med Genet. 1991; 39: 472-473Crossref PubMed Google Scholar) and repetitive sequences such as DXZ1 and DYZ1.6Levinson G Keyvanfar K Wu JC Fugger EF Fields RA Harton GL Palmer FT Sisson ME Starr KM Dennison-Lagos L Calvo L Sherins RJ Bick D Schulman JD Black SH DNA-based X-enriched sperm separation as an adjunct to preimplantation genetic testing for the prevention of X-linked disease.Hum Reprod. 1995; 10: 979-982PubMed Google Scholar, 7Hashiba T Sueoka K Kuroshima M Asada H Kuji N Yoshimura Y Accurate multiplex polymerase chain reaction assay for gender determination from a single cell.Gynecol Obstet Invest. 2000; 49: 217-220Crossref PubMed Google Scholar Sequences common to the sex chromosomes are identical at the site of primer annealing but differ internally in terms of size or include minor polymorphisms. Despite these technical improvements, the majority of embryo sexing is now accomplished using fluorescent in situ hybridization (FISH) which is less prone to contamination and can also provide the copy number for each chromosome tested thereby potentially avoiding the transfer of common chromosome abnormalities such as triploidy or X-monosomy.8Griffin DK Wilton LJ Handyside AH Atkinson GH Winston RM Delhanty JD Diagnosis of sex in preimplantation embryos by fluorescent in situ hybridisation.Br Med J. 1993; 306: 1382Crossref PubMed Google Scholar, 9Grifo JA Tang YX Munne S Alikani M Cohen J Rosenwaks Z Healthy deliveries from biopsied human embryos.Hum Reprod. 1994; 9: 912-916PubMed Google Scholar Although FISH has largely superseded PCR for sex determination, the specific diagnosis of single-gene defects remains dependent on DNA amplification with PCR. In the case of X-linked disorders, testing of the specific gene has the added advantage of ensuring that all embryos free of the mutant gene can be selected for transfer, irrespective of gender.10Liu J Lissens W Van Broeckhoven C Lofgren A Camus M Liebaers I Van Steirteghem A Normal pregnancy after preimplantation DNA diagnosis of a dystrophin gene deletion.Prenat Diagn. 1995; 15: 351-358Crossref PubMed Google Scholar, 11Hussey ND Donggui H Froiland DA Hussey DJ Haan EA Matthews CD Craig JE Analysis of five Duchenne muscular dystrophy exons and gender determination using conventional duplex polymerase chain reaction on single cells.Mol Hum Reprod. 1999; 5: 1089-1094Crossref PubMed Scopus (23) Google Scholar, 12Ray PF Vekemans M Munnich A Single cell multiplex PCR amplification of five dystrophin gene exons combined with gender determination.Mol Hum Reprod. 2001; 7: 489-494Crossref PubMed Google Scholar The list of disorders and the particular mutation detection strategies used for PCR-based clinical PGD application are given in Table 1. Essentially there are two laboratory components involved in PGD. The first involves the collection of diagnostic material for testing. This is usually performed in a clinical in vitrofertilization (IVF) laboratory under sterile conditions. A set of micromanipulators linked to an inverted microscope with contrast optics and facilities for extended embryo culture are the minimum essential requirements to carry out diagnostic biopsy procedures. The second step involves the diagnostic test itself, which can be performed in a region of the IVF laboratory, an adjacent laboratory equipped to perform molecular analyses or in a completely separate dedicated molecular genetics laboratory equipped to process single cell samples. Minimum requirements include a PCR preparation area (usually a small, dedicated flow hood), dedicated PGD reagent storage facilities, thermal cycler, and access to the necessary post-PCR mutation detection apparatus. The critical component of the diagnostic step is to minimize the level of contamination and a number of possible laboratory designs and procedures may fulfill this requirement. Theoretically, diagnostic material can be collected at any developmental stage between the mature oocyte and blastocyst. To date, four distinct stages have been targeted; metaphase II oocyte, zygote, cleavage stage embryo, and blastocyst. The four stages dictate different diagnostic strategies, each with its own limitations. The different technical approaches required to obtain the material and the material itself can affect the success rate of the procedure. The strengths and limitations of each approach are summarized in Table 2.Table 2Strategic Considerations for PCR Analysis of Diagnostic Material Biopsied at Different Developmental Stages for Preimplantation Genetic DiagnosisStageAdvantagesDisadvantagesOocyte (1st polar body)Removal has no effect on embryo development Increased time to perform PCR analysis prior to transferOnly 1 cell available for analysis Increased risk of diagnostic error Maternally inherited disease only Fewer embryos for transfer (recombination)Zygote (1st and 2nd polar body)2 cells for analysis (greater accuracy/reliability) Removal has no effect on embryo development Increased time to perform PCR analysis prior to transferMaternally inherited disease only Narrow time window to complete biopsyCleavage stage (blastomeres)Diagnosis of maternally/paternally inherited disorders Large body of clinical data available 2 cells available for analysis (greater accuracy/reliability)Chromosomal mosaicism present Selection of nucleated blastomere is criticalBlastocyst (trophectoderm)Sample multiple cells (eliminate PCR failure/ADO) Trophectoderm sampled rather than inner cell mass Embryo quality preselected Higher implantation rate/lower multiple gestation rateTime for PCR analysis may be limited Cells may not be representative of embryo Fewer embryos for analysis Limited clinical data available Open table in a new tab Each of the biopsy methods involves at least two steps; the first step being to breach the zona pellucida while the second involves the removal of cellular material (be that polar body, blastomere, or trophectodermal cells). Zona breaching can be achieved mechanically (by means of a sharp microneedle), chemically (using acidified Tyrodes solution, pH 2.2), or by thermal ablation (using a non-contact laser). Removal of cellular material is generally carried out using a glass micropipette attached to a pneumatic or hydraulic based suction system.13Harper JC Thornhill AR Embryo biopsy.in: Harper JC Delahanty JDA Handyside AH Preimplantation Genetic Diagnosis. John Wiley and Sons, Chicester UK2001: 141-163Crossref Google Scholar At present, polar body biopsy in combination with PCR based assays is performed almost exclusively by one group14Verlinsky Y Ginsberg N Lifchez A Valle J Moise J Strom CM Analysis of the first polar body: preconception genetic diagnosis.Hum Reprod. 1990; 5: 826-829Crossref PubMed Google Scholar, 15Verlinsky Y Rechitsky S Cieslak J Ivakhnenko V Wolf G Lifchez A Kaplan B Moise J Walle J White M Ginsberg N Strom C Kuliev A Preimplantation diagnosis of single gene disorders by two-step oocyte genetic analysis using first and second polar body.Biochem Mol Med. 1997; 62: 182-187Crossref PubMed Scopus (51) Google Scholar while the majority of PGD centers16ESHRE PGD Consortium Steering Committee ESHRE Preimplantation Genetic Diagnosis (PGD) Consortium: preliminary assessment of data from January 1997 to September 1998.Hum Reprod. 1999; 14: 3138-3148Crossref PubMed Google Scholar obtain genetic material for PGD by cleavage stage biopsy on the third day following insemination when the embryo has between 6 and 10 cells. At this stage, blastomeres are believed

Referência(s)