Hum. Reprod. Advance Access originally published online on June 3, 2008
Human Reproduction 2008 23(8):1949-1956; doi:10.1093/humrep/den201
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Birth of a healthy boy after a double factor PGD in a couple carrying a genetic disease and at risk for aneuploidy: Case Report
1 Unitat de Biologia Cel·lular i Genètica Mèdica, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Spain 2 Laboratoria de FIV, Fundación Jiménez Díaz, Plaza Reyes Católicos 2, Madrid, Spain 3 Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Oxford, UK
4 Correspondence address. Tel: +34-93-581-1773; Fax: +34-93-581-1025; E-mail: joaquima.navarro{at}uab.es
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsGene analysis approachPGD clinical caseResultsDiscussionFundingReferences |
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Preimplantation genetic diagnosis (PGD) for monogenic diseases is widely applied, allowing the transfer to the uterus of healthy embryos. PGD is also employed for the detection of chromosome abnormalities for couples at high risk of producing aneuploid embryos, such as advanced maternal (>35 years). A significant number of patients requesting PGD for monogenic diseases are also indicated for chromosome testing. We optimized and clinically applied a PGD protocol permitting both cytogenetic and molecular genetic analysis. A couple, carriers of two cystic fibrosis (CF) mutations (c.3849 + 10 KbC > T and c.3408C > A) with a maternal age of 38 years and two previously failed IVF–PGD cycles, was enrolled in the study. After ovarian stimulation, six oocytes were obtained. To detect abnormalities for all 23 chromosomes of the oocyte, the first polar body (1PB) was biopsied from five of the oocytes and analyzed using comparative genomic hybridization (CGH). CGH analysis showed that 1PB 1 and 1PB 4 were aneuploid (22X,–9,–13,+19 and 22X,–6, respectively), while 1PB 2, 1PB 3 and 1PB 6 were euploid. Blastomere biopsy was only applicable on embryos formed from Oocyte 3 and Oocyte 6. After whole-genome amplification with multiple displacement amplification, a multiplex PCR, amplifying informative short tandem repeats (D7S1799; D7S1817) and DNA fragments encompassing the mutation sites, was performed. MiniSequencing was applied to directly detect each mutation. Genetic diagnosis showed that Embryo 6 was affected by CF and Embryo 3 carried only the c.3849 + 10 KbC > T mutation. Embryo 3 was transferred achieving pregnancy and a healthy boy was born. This strategy may lead to increased pregnancy rates by allowing preferential transfer of euploid embryos.
Key words: PGD/cystic fibrosis/aneuploidy/PCR/comparative genomic hybridization
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Introduction |
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TopAbstractIntroductionMaterials and MethodsGene analysis approachPGD clinical caseResultsDiscussionFundingReferences |
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Preimplantation genetic screening (PGS) to detect chromosomal abnormalities has been applied to the patients of advanced maternal age (AMA), with recurrent spontaneous abortions, recurrent IVF failure or severe male factor, in order to identify aneuploid embryos. A negative selection against chromosomal abnormalities during the first stages of embryonic development is present (Boue et al., 1985
) and the consequent embryonic wastage is probably one of the main factors contributing to the low fertility rate in humans (Bahce et al., 1999; Sandalinas et al., 2001). It has been suggested that if embryos found to be euploid are prioritized for transfer during IVF cycles, pregnancy rates should be increased and spontaneous abortion rates decreased (Gianaroli et al., 2002; Munne et al., 2006; Farfalli et al., 2007).
In most cases, PGS involves cytogenetic analysis of single blastomeres biopsied 3 days after fertilization or polar bodies (PBs) biopsied prior to the first mitotic division. Generally, chromosomes are assessed using fluorescent in situ hybridization (FISH). However, according to the European Society of Human Reproduction and Embryology (ESHRE) preimplantation genetic diagnosis (PGD) Consortium data collection I–VI (Sermon et al., 2007), only in 15% (1013/6737) of the transferred embryos selected by PGS implant, as indicated by a positive HCG, and just 13.6% (918/6737) result in a pregnancy with detection of a fetal heartbeat. Different groups have found no difference or even lower implantation rate when comparing patients treated with PGS with control IVF patients (Staessen et al., 2004; Mastenbroek et al., 2007). In our opinion, the main limitation of PGS using FISH is the limited number of chromosomes that can be analyzed (9–13 probes in two rounds of FISH) (Abdelhadi et al., 2003; Pujol et al., 2003), which means that 43–60% of chromosomes are not analyzed. In order to achieve a significant improvement in implantation rates per embryo transfer, full karyotype analysis would be recommended (Wells and Delhanty, 2000).
Comparative genomic hybridization (CGH) is a molecular cytogenetic technique that allows the analysis of the full set of chromosomes by co-hybridization of a euploid reference DNA fluorescently labeled (in green) and a test DNA labeled with another fluorochrome (in red) to euploid metaphase spreads (Kallioniemi et al., 1992). CGH has been optimized to be used on single cells (Voullaire et al., 1999, 2000; Wells et al., 1999; Wells and Delhanty, 2000; Gutierrez-Mateo et al., 2004a,b). Not only does CGH permit screening of the entire chromosome complement, but it also eliminates the need to spread the biopsied cell on a microscope slide, since single cells are placed intact into PCR tubes. Fixation and spreading of single cells is technically challenging, sometimes resulting in artefactual chromosome losses (Fragouli et al., 2006a,b).
So far, two approaches for PGS employing CGH analysis have been developed. The first involves the application of CGH to a single blastomere. As there is not enough time to obtain the CGH result in the same IVF cycle (CGH requires 4 days to obtain results), embryo freezing is necessary (Voullaire et al., 2002). Unfortunately, this strategy is problematic due to the fact that about 33–50% of the embryos do not survive the freezing–thawing process depending on the used protocol (Hill, 2003; Jericho et al., 2003; Munne and Wells, 2003; Verlinsky and Kuliev, 2003; Wilton et al., 2003).The second approach is to apply CGH to the first polar body (1PB). Since the 1PB is biopsied immediately after fertilization by ICSI on Day 0 (Durban et al., 2001), CGH results can be obtained on Day +3 or +4 allowing for the transfer of embryos derived from cytogenetically normal oocytes to the maternal uterus, on Day +4 or +5, without the need for cryopreservation (Wells et al., 2002).
CGH analysis of the 1PB permits indirect cytogenetic characterization of the corresponding oocyte. This, in turn, allows for the detection of aneuploidies resulting from abnormal meiosis I segregation, which are frequently observed in first-trimester spontaneous abortions (Nicolaidis and Petersen, 1998; Hassold and Hunt, 2001). CGH-1PB has been successfully applied for PGS (Wells et al., 2002). In a research context, many 1PBs and metaphase II oocytes have been analyzed by CGH, revealing an aneuploidy frequency ranging from 22 to 53% (Gutierrez-Mateo et al., 2004a,b; Fragouli et al., 2006a,b).
Another option in order to perform a full karyotype analysis is the application of CGH-array technology to either blastomeres or 1PBs (Hu et al., 2004; Wells et al., 2004; Le Caignec et al., 2006). This methodology has huge potential having all the benefits of conventional CGH, but being much quicker to perform and less labor intensive. However, a few studies have assessed the reliability of this approach using single cells and there are no published reports of clinical application.
PGD for monogenic diseases has been applied extensively on over 1200 clinical cases, as has been stated in the ESHRE PGD Consortium data collection I–VI (Sermon et al., 2007). In order to select embryos free of the causative mutation, many PCR-based PGD protocols have been described, employing a wide range of methods for mutation detection (e.g. restriction digestion, variation of electrophoretic mobility by single-stranded conformation polymorphism or denaturing gradient gel electrophoresis and ‘MiniSequencing’) (Vrettou et al., 1999; Piyamongkol et al., 2001a; Abou-Sleiman et al., 2002b; Bermudez et al., 2003).
‘MiniSequencing’ is a versatile method allowing identification of many specific mutations (Fiorentino et al., 2003). Basically, a specific primer is designed to anneal directly adjacent to the mutation site and a single fluorescent dideoxynucleotide (ddNTPs) complementary to the wild-type/mutation nucleotide in the template is added (i.e. primer extension reaction is performed). This process, which is repeated in successive rounds of extension and termination by PCR, generates fluorescent-labeled fragments that are analyzed by capillary electrophoresis. Each of the four possible ddNTPs is labeled with a different color, allowing the sequence of the template to be deduced. ‘MiniSequencing’ is a highly sensitive technique, capable of detecting multiple point mutations at once.
Furthermore, indirect diagnosis using linked short tandem repeats (STRs) to detect the haplotype associated with the mutated gene decrease the risk of misdiagnosis in PGD (Piyamongkol et al., 2001b; Spits et al., 2005).
Most PGD strategies use direct amplification and analysis of DNA from blastomeres. However, an alternative is to employ a more generalized amplification prior to amplification of individual loci (Ao et al., 1998). Whole-genome amplification of the cell with multiple displacement amplification (MDA) prior to PCR amplification has been previously used (Hellani et al., 2004, 2005; Lledo et al., 2006), retrieving enough DNA from one single cell to amplify up to 64 loci (Renwick et al., 2007).
Even in women younger than 35 years involved in PGD for monogenic disease, the implantation rate is, according to the ESHRE PGD Consortium data collection I–VI, only 15.9% (405/2543 HCG positive) and only 9% of the transferred embryos (230/2543) produced a pregnancy, as determined by detection of a fetal heartbeat (Sermon et al., 2007). Since cytogenetic abnormalities could contribute to the low implantation rate, a PGD strategy accounting for both forms of genetic risk (monogenic disease and aneuploidy in embryos) would be beneficial. The aim of this work was to optimize a PGD procedure combining aneuploidy screening of the retrieved oocytes, achieved using CGH of 1PB, and monogenic disease detection using both ‘MiniSequencing’ and linkage analysis of biopsied blastomeres.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsGene analysis approachPGD clinical caseResultsDiscussionFundingReferences |
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In a carrier couple of cystic fibrosis (CF[MIM 219700 [OMIM] ]) with an affected child, causative mutations in the CF transmembrane regulator (CFTR) gene were c.3849 + 10 KbC > T and c.3408C > A, for the female and the male, respectively. Moreover, the female was 38 years old and had undergone two previous IVF–PGD cycles without achieving pregnancy, although an embryo transfer was only performed in one of the IVF cycles. Hence, the family had two factors risks; a risk of CF and an aneuploidy risk due to AMA. For this reason, a double factor PGD (DF-PGD) was recommended.
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Gene analysis approach |
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TopAbstractIntroductionMaterials and MethodsGene analysis approachPGD clinical caseResultsDiscussionFundingReferences |
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Mutation detection in genomic DNA
Outer and inner pairs of primers (forward and reverse) for nested-PCR amplification of sequences encompassing each mutation were designed (Primer3, http://frodo.wi.mit.edu) and acquired. Two STRs close to the CFTR gene, D7S1799 and D7S1817, were chosen according to the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?taxid=9606). Forward primers for these STRs were labeled with 6FAM and PET dyes, respectively (Table I). All primers were obtained from Roche Applied Science (Basel, Switzerland).
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A multiplex PCR containing outer primers for each mutation site and labeled primers for the two STRs was performed. The reaction mix contained the primer volumes indicated in Table I, 0.5 µl of genomic DNA, 2.5 µl of a 2 mM dNTPs Mix, 1x HotMaster Buffer and 1 U HotMaster Taq Polymerase (Eppendorf, Hamburg, Germany) in a final volume of 25 µl. A first round of DNA amplification was performed in a thermocycler (TGradient, Biometra, Goettingen, Germany) using the following PCR protocol: 2 min at 94°C, 37 cycles of 20 s at 94°C, 45 s at 55°C and 30 s at 65°C, and finally 5 min at 65°C. The alleles of both STRs were detected by analyzing 1 µl of the product in an ABIPrism 370 sequencer (AppliedBiosystems, CA, USA).
A second-round multiplex was performed with 0.5 µl of the product of the first-round multiplex mix as a template DNA. The reaction mix contained the inner primers for both mutations in volumes indicated in Table I and 2.5 µl of a 2 mM dNTPs Mix, 1x HotMaster Buffer and 1 U HotMaster Taq Polymerase (Eppendorf) in a final volume of 25 µl.
The second round of DNA amplification was performed in a thermocycler (TGradient, Biometra) using the following PCR protocol: 2 min at 94°C, 30 cycles of 20 s at 94°C, 45 s at 57°C and 30 s at 65°C, and then 5 min at 65°C. The amplification of the inner fragments of both mutations was verified by agarose gel electrophoresis.
Direct mutation detection was performed by ‘MiniSequencing’, following the manufacturer's instructions (Snapshot Multiplex Kit, AppliedBiosystems). Reverse inner primers of both mutations were designed to be used as ‘MiniSequencing’ primers. Because these primers bind to the antisense DNA strand the nucleotide change detected for the c.3849 + 10 KbC > T mutation will be G to A and for the C.3408C > A mutation will be G to T instead of the alteration mentioned above. The presence or absence of both mutations was detected by analyzing 1 µl of ‘MiniSequencing’ product in an ABIPrism 3730 sequencer (Applied Biosystems, CA, USA).
Gene analyses in single-cell DNA
Prior to PGD, a total of 30 single buccal cells were used in the following protocol. Whole-genome amplification was performed on each single cell using the MDA technique using Genomiphi v2 DNA Amplification Kit (GE Healthcare, Buckinghamshire, UK). Some slight modifications have been introduced from the recommended manufacturer protocol in order to perform the single cell lysis. Briefly, 2.5 µl Alkaline Buffer (ALB; 200 mM KOH and 50 mM dithiothreitol) and 5 µl of sample buffer were added to each 0.2 ml PCR tube containing a single buccal cell. The samples were kept 3 min at 95°C using a Thermocycler (TGradient, Biometra) and immediately put on ice. To neutralize the ALB, 0.8 µl of Tricine (20 mM, PH 4.95) was added (Spits et al., 2006). Then, 8.2 µl of Reaction Buffer and 1 µl of enzyme mix provided in the kit were added. The MDA reaction took place for 90 min at 30°C and 10 min at 70°C. Four microliters of the MDA-amplified cell product was used as a genomic DNA template and the genetic analysis was performed as has been described above.
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PGD clinical case |
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IVF procedure and biopsy of 1PB and blastomere
The female of the couple underwent a routine superovulation procedure. The cells of the cumulus and corona radiata were completely removed by a combination of an enzymatic and mechanical procedure. The 1PB biopsy was performed on Day 0, as previously described (Durban et al., 2001
). Briefly, the 1PB was removed by the partial zone dissection (PZD) procedure using a thin and sharp glass micropipette (PZD Micropipette, Humagen, VA, USA) to create a hole in the zone pellucida by mechanical force (Malter, 1989). The 1PB biopsy was performed immediately after the corresponding oocyte was inseminated by ICSI. The 1PB was aspirated using a 13–15 µm internal diameter (ID) micropipette (MPB-BP-30 Micropipette, Humagen), washed four times with different, sterile phosphate-buffered saline (PBS)/0.1% polyvinyl alcohol (PVA) droplets and put into a labeled 0.2 ml PCR tube and kept at –20°C. The 1PB biopsied on Day 0 was shipped from the IVF laboratory in Madrid to our center at the Autonomous University of Barcelona in Bellaterra on dry ice 20 h after the follicular puncture. On Day +3 after ICSI, one blastomere was biopsied from the 6–8 cell stage embryos using the same hole created previously in the 1PB removal and using a 35 µm ID micropipette (MBB-BP-M-30 Micropipette, Humagen). Each blastomere was washed in PBS/0.1% PVA solution and was put into a labeled 0.2 ml PCR and kept at –20°C.
Genetic analysis of the blastomere
Direct and indirect mutation analysis of each blastomere was performed in a product of MDA as described above.
CGH analysis in 1PB
To each 0.2 ml PCR-labeled tube containing 1PB, 1 µl of sodium dodecyl sulfate (17 µM) and 2 µl of proteinase K (125 µg/ml) were added. The lysis was performed by incubating at 37°C for 1 h followed by 10 min at 95°C to inactivate proteinase K, using a thermocycler (TGradient, Biometra). After that, whole-genome amplification by degenerate oligonucleotide-primed PCR was applied to the samples as previously described (Gutierrez-Mateo et al., 2004b) in order to increase the amount of DNA. For use as test DNA, amplified 1PBs were fluorescently labeled with Spectrum Red-dUTP (Vysis, Downers Grove, USA) by Nick Translation (Vysis) following the manufacturer's instructions. As reference DNA, three single, buccal epithelium cells from a euploid woman (46,XX) were isolated and treated like the 1PBs, but labeled with Spectrum Green-dUTP (Vysis). Precipitation of DNA and hybridization over euploid male metaphase spreads was performed as previously described (Gutierrez-Mateo et al., 2004a,b), but hybridization in a moist chamber at 37°C was for 44 h instead of 72 h.
At least 10 metaphases per 1PB-CGH were captured with an epifluorescence microscope by SmartCapture software (Digital Scientific, Cambridge, UK) and karyotyped by Vysis Quips CGH software (Vysis).
When the fluorescence ratio (test/reference) described by the software is <0.8, a chromosome loss is present in the DNA test, whereas when the ratio is >1.2, a chromosome gain is present (Wells et al., 1999).
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Results |
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Results from genomic DNA
The STRs are shown in Table II. The STR D7S1799 is informative for the maternal mutation (c.3849 + 10 KbC > T), while D7S1817 is informative for the paternal mutation (c.3408C > A).
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Mutations c.3849 + 10 KbC > T and c.3408C > A were directly detected by ‘MiniSequencing’ as expected. In the DNA of the mother, a blue peak corresponding to the wild-type (Guanine) and a green peak corresponding to the mutated nucleotide (Adenine) were observed. In the DNA of the father, a blue peak to the wild-type allele (Guanine) and a red peak corresponding to the mutated nucleotide (Thymine) were obtained. In the DNA of the CF-affected son, both of the peaks, wild-type and mutated, corresponding to both parental mutations, were observed (Fig. 1).
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STR and mutation results in single-cell MDA product
DNA from 30 isolated single cells from the affected member of the family was successfully amplified with MDA (30/30, 100%) and treated as described. The expected results for the corresponding informative STR were observed in 26/30 single cells for D7S1799, and in 22/30 single cells for D7S1817, indicating an allele drop-out (ADO) rate of 13 and 26%, respectively. Moreover, expected results for the corresponding mutation in the ‘MiniSequencing’ electropherograms were obtained in 19/30 cells for the c.3849 + 10 KbC > T mutation and in 22/30 cells for the c.3408C > A mutation of the analyzed cells, indicating an ADO rate of 36 and 26%, respectively.
In-vitro fertilization
In all cumulus–oocyte complexes retrieved, the corresponding 1PB was obtained following ICSI on Day 0, with the exception of Oocyte 5, which had no 1PB. On Day +1 after follicular puncture, fertilization was confirmed by identification of two pronuclei in zygotes 1, 3 and 6. On Day +3, only Embryos 3 and 6 achieved 6- to 8-cell stage and showed a good embryo quality. Therefore, only one blastomere from Embryos 3 and 6 was biopsied and subjected to testing.
Cytogenetic analysis in 1PB
All 1PBs biopsied and analyzed by CGH gave good results (Fig. 2). Two of the five 1PBs were aneuploid for one or more chromosomes. The 1PB 1 had a CGH profile corresponding to losses of chromosomes 9 and 13 and a gain of chromosome 19. An embryo derived from this oocyte would be at risk of trisomy for chromosomes 9 and 13 and monosomy for 19. 1PB 4 showed a loss of chromosome 6, which would lead to a chromosome 6 gained in the oocyte and, hence, a risk of trisomy 6. 1PB 2, 1PB 3 and 1PB 6 were totally euploid for the 23 chromosomes (Table II).
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STR and mutation analysis in blastomeres
Concordant results were obtained by STR analysis and the direct mutation detection analysis in the product of MDA from the blastomere of Embryos 3 and 6. Although Embryo 3 was transferable, being the carrier of the c.3408C > A paternal mutation, Embryo 6 was affected by CF (Fig. 1 and Table II).
Final PGD outcome
Embryo 3 was transferred on Day (+4) achieving a pregnancy verified with a positive HGC value and fetal heartbeat. Prenatal diagnosis was performed by an external laboratory with quantitative fluorescent PCR and showed a normal copy-number of the analyzed chromosomes. A healthy boy, a carrier of the paternal mutation, has been delivered.
Re-analysis of rejected unfertilized oocytes or arrested embryos
Except for Oocyte 5, which did not display 1PB, the rest of the rejected materials (unfertilized Oocytes 2 and 4 and arrested Embryo 1) were processed for genetic analysis of the parental mutations, as previously described. Oocyte 2 was confirmed to be free of the maternal mutation. The arrested Embryo 1 was found to be a carrier of the paternal mutation and the maternal mutation was present in unfertilized Oocyte 4 (Table II).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsGene analysis approachPGD clinical caseResultsDiscussionFundingReferences |
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The strategy for the direct analysis by ‘MiniSequencing’ of parental mutations c.3849 + 10 KbC > T and c.3408C > A performed well, allowing accurate identification of the complementary wild-type and mutated nucleotides (see results in Table II). A similar approach has been successfully applied to a variety of other monogenic gene disease PGD protocols (Cram et al., 2003
; Fiorentino et al., 2003; Iacobelli et al., 2003).
Additionally, indirect detection of parental mutations using closely linked tetra-nucleotide STRs proved to be extremely useful for the detection of mutant or wild-type alleles, significantly improving diagnostic accuracy. The use of two independent mutation-detection approaches at the same time, direct and indirect mutation analysis, is a very convenient and safe approach and it is widely applied by different groups currently performing PGD (Abou-Sleiman et al., 2002a; De Rycke et al., 2005; Kuliev et al., 2006).
Whole-genome amplification of single cells using MDA was applied. MDA is a very useful procedure that produces a large amount of high-quality DNA. The PCR multiplex, used for the simultaneous amplification of the two mutation sites and two linked polymorphisms, displayed superior performance when applied to MDA products compared with direct application to single cells (Hellani et al., 2004, 2005; Burlet et al., 2006; Lledo et al., 2006; Coskun and Alsmadi, 2007). No differences in the size (bp) of the peaks were observed in the three genomic DNAs compared with those observed after MDA of a single cell when indirect or direct genetic analysis was performed (Table II).
The ADO rates observed during pre-clinical validation of the PGD test (13% for D7S1799 and 26% for D7S1817 following MDA of single buccal cells) are similar to the 28% described after analyzing 64 loci of 49 blastomeres in a previous study (Renwick et al., 2007). In contrast, the ADO observed in one of the mutation sites was higher (36% of all cells for the c.3849 + 10 KbC > T mutation). This may be explained by the fact that buccal cells are arrested in the G0 stage and the apoptotic process can be present. Consequently, the DNA produced is likely to be of reduced quality compared with fresh proliferative cells, as was the case for single blastomeres. Despite relatively high ADO rates, the risk of misdiagnosis of an affected embryo for a healthy embryo, due to ADO affecting the mutant allele for all four loci in the same cell, is estimated to be much lower than 0.02%. The low error rate is a consequence of assessing multiple diagnostically relevant markers (i.e. mutation sites and also linked markers). ADO is highly unlikely to simultaneously affect all diagnostic loci. In fact, the higher ADO rates observed in MDA products compared to conventional single-cell PCR is one of the main concerns of the MDA approach, a fact that could lead to a misdiagnosis on PGD. However, as demonstrated here, the ability to analyze multiple loci simultaneously permits a low misdiagnosis rate despite increased ADO. Using MDA, multiple independent PCR amplifications can be undertaken, allowing examination of multiple loci, while avoiding difficulties associated with multiplexing of PCR primers.
For the clinical PGD case, no ADO was observed for either the STR or the ‘MiniSequencing’ products of the blastomeres studied. Although the number of analyzed cells was small, this observation may indicate that proliferative cells are more appropriate for evaluating ADO rates than buccal cells, as stated above.
Informative results were obtained in five of the six mature oocytes that were cytogenetically analyzed during PGD by CGH, using a modified version of the protocol employed previously by our group (Gutierrez-Mateo et al., 2004b). Two of the five (40% of the analyzed oocytes) displayed abnormalities involving one or more chromosomes, the rest of the oocytes were euploid. The frequency of aneuploidy observed is in accordance with the frequencies described in different published works using CGH from [22% (Fragouli et al., 2006b) to 52% (Gutierrez-Mateo et al., 2004a,b)].The chromosomes involved in the aneuploidies observed in the oocytes of the current study (6, 9, 13 and 19) have been previously observed in different studies using FISH or CGH analysis of oocytes and PBs (Anahory et al., 2003; Cupisti et al., 2003; Gutierrez-Mateo et al., 2004a; Pujol et al., 2006).
One of the abnormal oocytes (4) would not have been identified as being aneuploid by conventional FISH analysis due to the fact that chromosome 6 is not included in the panels of FISH probes typically employed for the purposes of PGD/PGS. In order to accurately detect the copy number of all chromosomes, PGS by CGH-1PB analysis is an attractive alternative to PGS by FISH.
Considering the genetic results obtained in the direct and indirect analysis of the mutation in the two developing embryos, only Embryo 3 was found to be healthy (carrier of the paternal mutation). The other developing Embryo 6 was revealed to be affected by CF and was rejected for transfer. Re-analysis of Embryo 6 confirmed the result obtained in the initial PGD. Combining this genetic result of Embryo 3 with the CGH result for PB 3, a healthy euploid embryo was expected. After Genetic Counseling given to the family, Embryo 3 was transferred and a pregnancy was achieved. Prenatal diagnosis showed a chromosomally normal fetus and a healthy boy, carrier of the paternal CF mutation, has since been delivered.
The DF-PGD procedure applied in the present case had a satisfactory outcome. The analysis of the 1PB by CGH allowed comprehensive chromosomal screening, while avoiding the need for embryo freezing. It may also be worth considering the use of chromosome screening in PGD cycles in which the maternal age is not a risk for aneuploidy. Patients undergoing PGD of monogenic diseases are often of relatively young maternal age (33 years), yet implantation rates are only 15.6% (94/603) and 12.4% (75/603) as detected by positive HCG or positive heartbeat (Sermon et al., 2007). The complete cytogenetic analysis of the oocytes may be a very good approach in order to increase the pregnancy rate, even in the case of young women, as these patients also have an appreciable aneuploidy rate in their oocytes, albeit significantly lower than older patients (Sher et al., 2007).
In conclusion, in the present work a successful procedure combining comprehensive analysis of aneuploidy in 1PB with detection of two infrequent mutations in the CFTR gene (c.3408C > A and c.3846 + 10KbC > T) has been achieved for the first time.
The combination of chromosome screening using CGH analysis and single gene testing requires a highly coordinated and multidisciplinary team. In spite of the complexity of techniques needed and logistical difficulties in undertaking CGH in a fresh cycle, we believe this is a worthwhile challenge potentially increasing implantation rates for cycles of PGD for single gene disorders.
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Funding |
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This research study was been funded by Ministerio de Sanidad y Consumo Fondo de Investigación Sanitaria Instituto de Salud Carlos III (FIS-ISCIII; PI 051395) and by Grup de Suport a la Recerca. Generalitat de Catalunya 2005SGR00 495. Albert Obradors has a predoctoral grant of the Generalitat de Catalunya (2005FI00 108).
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References |
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Submitted on January 18, 2008; resubmitted on March 31, 2008; accepted on April 17, 2008.
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