Preimplantation Genetic Diagnosis Cases Research Articles

Development of a PCR-based method to identify fetal sex during IVF cycles.

One of the most recognizable cases of preimplantation genetic diagnosis (PGD) is X-linked diseases. Diagnosis of fetal sex is essential for couples who are known to be at risk of some X-linked disorders. The objective of this study was to discriminate between female (XX) and male (XY) embryos by detecting sex chromosomes-specific sequences in spent culture medium and comparing these results to PGD/CGH array results. It may open new window for the development of a non-invasive PGD method. 120 Embryo's spent media from Day 3 and Day 5 embryos were collected. Modified phenol-chloroform solution was used for DNA extraction from spent media. Sex determination was performed using SRY, TSPY and AMELOGENIN evaluation through quantitative polymerase chain reaction (q-PCR) method. IBM SPSS and MedCalc were used for statistical analyses to compare sex determination of embryos by spent medium with PGD/CGH array results. Culture time was demonstrated to increase the DNA amount among day 5 embryos culture medium samples. Non-invasive PGD by means of spent culture medium gave a sensitivity, specificity, positive predictive value and negative predictive value of 100% for sex determination. Results of sex determination using spent medium by q-PCR were consistent with the results of PGD/CGH array. Improvements in cell-free DNA extraction and PCR amplification procedures provide us an effective method to perform a PGD test without biopsy in the future, especially about X-linked diseases.

Development and validation of a novel panel of 16 STR markers for simultaneous diagnosis of \u03b2-thalassemia, aneuploidy screening, maternal cell contamination detection and fetal sample authenticity in PND and PGD/PGS cases

Prenatal diagnosis (PND) may be complicated with sample mix-up; maternal cell contamination, non-paternity and allele drop out at different stages of diagnosis. Aneuploidy screening if combined with PND for a given single gene disorder, can help to detect any common aneuploidy as well as aiding sample authenticity and other probable complications which may arise during such procedures. This study was carried out to evaluate the effectiveness of a novel panel of STR markers combined as a multiplex PCR kit (HapScreen™ kit) for the detection of β-thalassemia, aneuploidy screening, ruling in/out maternal cell contamination (MCC), and sample authenticity. The kit uses 7 STR markers linked to β-globin gene (HBB) as well as using 9 markers for quantitative analysis of chromosomes 21, 18, 13, X and Y. Selection of the markers was to do linkage analysis with β-globin gene, segregation analysis and to perform a preliminary aneuploidy screening of fetal samples respectively. These markers (linked to the β-globin gene) were tested on more than 2185 samples and showed high heterozygosity values (68.4–91.4%). From 2185 fetal cases we found 3 cases of non-paternity, 5 cases of MCC, one case of sample mix-up and one case of trisomy 21 which otherwise may have end up to misdiagnosis. This kit was also successfully used on 231 blastomeres for 29 cases of pre-implantation genetic diagnosis (PGD) and screening (PGS). The markers used for simultaneous analysis of haplotype segregation and aneuploidy screening proved to be very valuable to confirm results obtained from direct mutation detection methods (i.e. ARMS, MLPA and sequencing) and aneuploidy screening.

Clinical application of NGS-based SNP haplotyping for the preimplantation genetic diagnosis of primary open angle glaucoma

ABSTRACTThe current study describes a successful case of preimplantation genetic diagnosis (PGD) of primary open angle glaucoma (POAG) and verifies the efficiency of next-generation sequencing (NGS)-based haplotyping for PGD of POAG. In this study, we applied NGS as part of PGD to effectively detect POAG prior to embryo implantation and avoid the prospect of pregnancy termination in event of vertical inheritance of POAG. We used the technique of multiple annealing and looping based amplification cycles (MALBAC) to conduct whole genome amplification (WGA) and to reduce the allele dropout (ADO). We also employed Sanger sequencing to directly detect the mutation c.1109 C > T in MYOC and NGS-based single nucleotide polymorphism (SNP) haplotyping to distinguish the chromosomes that carried the mutation. Copy number variation (CNV) analysis was carried out to determine the copy number of embryos’ chromosomes. Of the 4 blastocysts obtained in this study, only 2 (sample 5 and 7) could be successfully amplified by WGA. CNV results indicated that chromosomes of both these samples were balanced (46, XN). Sanger sequencing and NGS-based SNP haplotyping confirmed that sample 7 carried the mutation c.1109 C > T in MYOC, while sample 5 did not. Moreover, no ADO was observed. Thus, blastocyst 5 was transferred into the uterus of the patient, and a healthy baby without the MYOC mutation c. 1109C>T was born 39 weeks after transplantation. Our study suggests that NGS-based SNP haplotyping is an effective technique for the PGD of POAG.Abbreviations: PGD: preimplantation genetic diagnosis; POAG: primary open angle glaucoma; NGS: next-generation sequencing; MALBAC: multiple annealing and looping based amplification cycles chemistry; WGA: whole genome amplification; ADO: allele dropout; SNP: single nucleotide polymorphism; CNV: copy number variation; MYOC: Myocilin; OPTN: Optineurin; WDR36: WD repeat domain 36; CYP1B1: Cytochrome P450 1 B Chain; ICSI: intracytoplasmic sperm injection; TFNA: testicular fine-needle aspiration; TE: trophectoderm; PCR: polymerase chain reaction

When standard genetic testing does not solve the mystery: a rare case of preimplantation genetic diagnosis for campomelic dysplasia in the setting of parental mosaicism

To report a rare case of somatic mosaicism with a germline component of campomelic dysplasia in a woman undergoing invitro fertilization with preimplantation genetic diagnosis (IVF-PGD). Case report. Clinic. A 28-year old G2P0110 and her 34-year old husband had two previous pregnancies complicated by fetal campomelic dysplasia with suspected germline mosaic mutation. The couple, both phenotypically normal, underwent IVF-PGD to reduce their chances of transmission. None of the embryos could initially be determined to be disease free, because all embryos shared either a maternal or a paternal short tandem repeat haplotype with the products of conception from her last pregnancy. Peripheral-blood cytogenomic single-nucleotide polymorphism (SNP) microarray to identify the carrier of the mutation, and IVF-PGD to identify the disease-free embryo. Disease-free embryo. Only one of the five euploid embryos was identified as disease free. A woman with suspected germline mosaicism for campomelic dysplasia was found to be a somatic mosaic with a germline component via a peripheral blood SNP microarray test. This identified her solitary disease-free embryo, which was transferred to her uterus but did not result in a viable pregnancy.

The first South American case of pre-implantation genetic diagnosis to select compatible embryo for cord blood transplantation as treatment for sickle cell anemia

Sickle cell anemia is an inherited systemic hemoglobinopathy that affects hemoglobin production in red blood cells, leading to early morbidity and mortality. It is caused by a homozygous nucleotide substitution (c.20A>T) in the β-globin gene (HBB) that changes a glutamic acid to a valine in the protein. We present a case report of a fertile couple, both carriers of the sickle cell anemia mutation, with one affected daughter. Six cycles of assisted reproductive techniques were performed, resulting in 53 embryos in cleavage stage. Each embryo was biopsied and analyzed for pre-implantation genetic diagnosis (PGD) by fluorescent polymerase chain reaction, using polymorphic markers of the region of interest followed by capillary electrophoresis in an automated genetic analyzer. HLA Compatible and normal embryos for the mutation represented 3 (5.66%); while the carriers and compatible 6 (11.32%); therefore, embryos matching those of the affected daughter represented 9 (16.98%). A selected embryo in blastocyst stage was transferred, resulting in a healthy male newborn, who had the umbilical cord blood cells collected and stored. The affected daughter was immunosuppressed and received transplanted cells from the umbilical cord blood of her brother; the treatment was successful. Embryo selection using PGD technologies represent the most effective treatment plan for parents who want to have a healthy child, and it could cure another child already affected by inherited hemoglobinopathy.

Pre-implantation genetic diagnosis and pre-implantation genetic screening: two years experience at a single center.

ObjectiveIndications for preimplantation genetic diagnosis (PGD)/preimplantation genetic screening (PGS) cycles and clinical outcomes were evaluated at CHA Gangnam Medical Center.MethodsThis is retrospective cohort study. All patients (n=336) who went through in vitro fertilization (IVF)-PGD/PGS cycles (n=486) between January 2014 and December 2015 were included in Fertility Center of CHA Gangnam Medical Center. Patients underwent IVF-PGD/PGS with 24-chromosome screening. Patients with euploid embryos had transfer of one or 2 embryos in a fresh cycle with any subsequent frozen embryo transfer (ET) cycle. Compared implantation, clinical pregnancy, ongoing pregnancy, and early abortion rates were the main outcome measures.ResultsThe most common indication for PGD/PGS was recurrent spontaneous abortion (n=160). The chromosome rearrangement cases (n=116) included 24 Robertsonian translocations, 60 reciprocal translocations, 3 inversions, 2 deletions, 4 additions, and 23 mosaicisms. PGS cases rather than the PGD cases showed higher implantation rates (26.4% vs. 20.3%), ongoing pregnancy rates (19.5% vs. 16.4%), and clinical pregnancy rates (28.6% vs. 23.3%). Implantation rates (30.3% vs. 23.7%), clinical pregnancy rates (39.2% vs. 25.2%), and ongoing pregnancy rates (25.7% vs. 17.5%) were significant higher in the blastocyst evaluation group than cleavage stage evaluation group.ConclusionThis was the largest study of PGD/PGS for 2 years at a single center in Korea. The pregnancy outcomes of PGD cases are slightly lower than PGS cases. It was confirmed again that success rate of PGD/PGS is higher if biopsy was done at blastocyst than cleavage stage.

Establishing a comprehensive genetic diagnosis strategy for hemophilia B and its application in Chinese population.

To reduce the incidence of hemophilia B (HB) which with no complete cure currently, prenatal diagnosis and preimplantation genetic diagnosis (PGD) are effective and feasible means. However, previous studies about genetic diagnosis in HB mostly just focused on the detection of patients and carriers. Here, we established a comprehensive genetic diagnosis strategy for HB and worked it out in Chinese population. The strategy includes the detection of patients and carriers, prenatal diagnosis, and PGD. Seven unrelated HB families from Chinese population involved in this study. Firstly, probands and available members were carried out coagulation laboratory assays, and the clinical information has been recorded. Secondly, we used DNA direct sequencing to screen the whole FIX gene of them. The pathogenicity of novel mutations was verified according to 2015 ACMG-AM guidelines. For prenatal diagnosis, a mix of DNA direct sequencing and STR linkage analysis was employed. To explore a better PGD protocol, Karyomapping was first applied in PGD of HB, comparing with conventional PCR-based methods. Six different pathogenic mutations including 1 novel duplication (c.660_661dup ATCA) were identified. The results of prenatal diagnosis were consistent with birth outcomes. In the PGD case, 4 of 11 embryos were confirmed to be normal and one of them was transferred and led to a healthy birth. The established genetic diagnosis strategy for HB in our study was comprehensive and well applied in clinic practice. Besides, we recommended that DNA direct sequencing combined with Karyomapping was a better PGD protocol.

Preimplantation genetic diagnosis (PGD) for borderline indications including human leukocyte antigens (HLA) matching, cancer predisposition, cardiac disease and their proportion in overall PGD cases for single gene disorders

Preimplantation genetic diagnosis (PGD) for borderline indications including human leukocyte antigens (HLA) matching, cancer predisposition, cardiac disease and their proportion in overall PGD cases for single gene disorders

Pre-implantation genetic diagnosis-should we use ICSI for all?

Intracytoplasmic sperm injection (ICSI) is commonly used during pre-implantation genetic diagnosis (PGD) in vitro fertilization (IVF), aiming to eliminate the risk of contamination from extraneous sperm DNA. Recently, ICSI "overuse" in non-male infertility has been doubted, since it does not offer an advantage over IVF. Prompted by the aforementioned observations, we sought to assess the accuracy of IVF vs ICSI in PGD cases, as might be reflected by a difference in the prevalence of discarded embryos as a consequent of parental contamination. Cohort-historical study of all consecutive patients admitted to the IVF-PGD program in a large tertiary center. The percentages of complete, incomplete diagnosis, PCR failure, abnormal embryos, and the contamination rate with paternal DNA in the IVF-only and the ICSI-only groups. We reviewed the computerized files of all consecutive women admitted to our IVF for a PGD-PCR cycle. Patients were divided accordingly into three groups: an IVF group-where all the oocytes underwent IVF only, an ICSI group-where all oocytes underwent ICSI, and a mixed group-where sibling oocytes underwent both IVF and ICSI. The laboratory data and the genetic diagnostic results were collected and compared between the different insemination groups. Nine-hundred and twenty-seven patients underwent IVF-PGD cycles in our program, 315 in the IVF group, 565 in the ICSI group, and 47 in the mixed group. No differences were observed in fertilization rates, the percentage of embryos available for biopsy, and the percentages of complete, incomplete diagnosis, PCR failure, or abnormal embryos, between the IVF-only and the ICSI-only groups and between the IVF and the ICSI of sibling oocytes in the mixed group. Moreover, contamination with paternal DNA, through contamination with sperm cells, was negligible. Not one single case of misdiagnosis was encountered during the study period. It might be therefore concluded that IVF should be the preferred insemination methods in PGD cycles, and ICSI should be indicated only in cases of male-factor infertility.

FMR1 CGG repeat expansion mutation detection and linked haplotype analysis for reliable and accurate preimplantation genetic diagnosis of fragile X syndrome.

Fragile X mental retardation 1 (FMR1) full-mutation expansion causes fragile X syndrome. Trans-generational fragile X syndrome transmission can be avoided by preimplantation genetic diagnosis (PGD). We describe a robust PGD strategy that can be applied to virtually any couple at risk of transmitting fragile X syndrome. This novel strategy utilises whole-genome amplification, followed by triplet-primed polymerase chain reaction (TP-PCR) for robust detection of expanded FMR1 alleles, in parallel with linked multi-marker haplotype analysis of 13 highly polymorphic microsatellite markers located within 1 Mb of the FMR1 CGG repeat, and the AMELX/Y dimorphism for gender identification. The assay was optimised and validated on single lymphoblasts isolated from fragile X reference cell lines, and applied to a simulated PGD case and a clinical in vitro fertilisation (IVF)-PGD case. In the simulated PGD case, definitive diagnosis of the expected results was achieved for all 'embryos'. In the clinical IVF-PGD case, delivery of a healthy baby girl was achieved after transfer of an expansion-negative blastocyst. FMR1 TP-PCR reliably detects presence of expansion mutations and obviates reliance on informative normal alleles for determining expansion status in female embryos. Together with multi-marker haplotyping and gender determination, misdiagnosis and diagnostic ambiguity due to allele dropout is minimised, and couple-specific assay customisation can be avoided.

PGD management scheme for older females with balanced translocations: Do older females have less chance of balanced embryo transfer?

Carriers of reciprocal and Robertsonian translocations have a higher risk of experiencing infertility and repeated miscarriages. It is well established that with advancing maternal age, the risk of aneuploidies in embryos increases. In this study, the chance of developing balanced embryos in translocation carriers with advanced maternal age was analyzed to establish a management scheme for couples seeking fertility treatment and preimplantation genetic diagnosis (PGD). Biopsy was performed on cleavage-stage embryos. Multicolor fluorescence in situ hybridization was used for PGD. The translocation carriers underwent a total of 55 cycles of PGD. Genetics diagnosis and cycle outcomes of PGD cases were examined. This study showed that the chance of obtaining a balanced embryo from the Robertsonian translocation carriers was significantly less when the maternal age is advanced. Similar rates for balanced embryos were obtained from the reciprocal translocation carriers. The results of this study show that maternal age plays an important role and that genetic counselling and planning for a PGD cycle in translocation carriers, particularly for Robertsonian carriers, must be accordingly adapted.

Current controversies in prenatal diagnosis 4: preimplantation genetic screening should be routinely offered to all preimplantation genetic diagnosis cases.

Current controversies in prenatal diagnosis 4: preimplantation genetic screening should be routinely offered to all preimplantation genetic diagnosis cases.

Live births following Karyomapping of human blastocysts: experience from clinical application of the method

The clinical application of a new, widely applicable method known as Karyomapping to carry out a total of 55 clinical cases of preimplantation genetic diagnosis (PGD) for single gene disorders is reported. Conventional polymerase chain reaction (PCR) testing was carried out in parallel to the new method for all cases. Clinical application of Karyomapping in this study resulted in three live births and nine clinical pregnancies out of 20 cases with a transfer. All in all, results presented in this study indicate that Karyomapping is a highly efficient, accurate and robust method for PGD of single gene disorders. Karyomapping can offer a more comprehensive assessment of the region of interest than conventional PCR analysis, allowing for more embryos to receive diagnosis (99.6% versus 96.8%), whereas its wide applicability reduces substantially the time that patients have to wait before starting their in vitro fertilization (IVF) cycle. Nonetheless, inclusion of elements of conventional PCR methodology, such as direct mutation detection, may be required in cases in which the gene of interest is in a region with reduced single nucleotide polymorphism (SNP) coverage (e.g. telomeric regions), when offering PGD for consanguineous couples, or in cases where no samples from additional family members are available.

Karyomapping-a comprehensive means of simultaneous monogenic and cytogenetic PGD: comparison with standard approaches in real time for Marfan syndrome.

Preimplantation genetic diagnosis (PGD) of single gene defects by genetic analysis of single or small numbers of cells biopsied from in vitro fertilization (IVF) embryos is clinically well-established. Targeted haplotyping by multiplex fluorescent polymerase chain reaction (PCR) of closely linked or intragenic short tandem repeat (STR) markers combined with direct mutation detection improves the accuracy of single cell analysis significantly and minimizes potential errors caused by undetected allele dropout (ADO) or contamination [1]. Allele dropout refers to the failure of one of the two alleles of a heterozygous locus to amplify. This makes a heterozygous cell appear homozygous at the affected locus, potentially leading to misdiagnosis. Furthermore, using high order multiplex protocols, this approach has been extended to multiple loci, including analysis of the Human leukocyte antigen (HLA) region for selection of embryos tissue matched to existing sick children and diagnosis of translocation chromosome imbalance [2–4]. However, the development of patient, disease or locus-specific protocols, and testing with single cells, is time-consuming and labour intensive. Also, this targeted approach only provides limited information on chromosome aneuploidy, which is recognized to be a major cause of IVF failure and pregnancy loss. As an alternative, we developed, “Karyomapping”—genome wide parental haplotyping using high density single nucleotide polymorphism (SNP) genotyping. Karyomapping provides a comprehensive method for linkage-based diagnosis of any single gene defect [5]. Genotyping of the parents and a close relative of known disease status, to phase informative SNP loci, eliminates the need for customized test development and, as Karyomapping defines four sets of SNP markers for each of the parental chromosomes, it allows simultaneous high-resolution molecular cytogenetic analysis. Thus, meiotic trisomies, including their parental origin, can be identified by the presence of both haplotypes from one parent in segments of the chromosome, resulting from the inheritance of two chromosomes with different patterns of recombination. Moreover, monosomies or deletions can be identified by the absence of either chromosome haplotype from the parent of origin [5]. Mitotic chromosome duplication, which can arise through malsegregation of chromosomes in the cleavage divisions following fertilization, cannot be detected by Karyomapping per se, since the sequence of both chromosomes is identical. However, chromosome duplications may be clinically less significant, since they are often associated with poor morphology and developmental arrest. In the past we have demonstrated that Karyomapping could be used for the detection of cystic fibrosis status in single cells [5]. Here we provide proof of principle for the widespread clinical application of Karyomapping, first by adapting the protocol for clinical use in a regular PGD timeframe (24 h) and secondly by detection of the autosomal dominant condition Marfan syndrome. Performing Karyomapping “as if in a clinical setting” for confirmation of results of an existing PGD case provides strong evidence of the applicability of Karyomapping and, in this case, led to a twin birth.

The probability of finding at least one euploid embryo based on retrieved oocytes in PGD cases for aneuploidy testing

Clinical success of Preimplantation Genetic Diagnosis (PGD) depends on many factors such as age and ovarian reserve of the patient. Although age has been extensively studied, probability of finding at least one euploid embryo (POFEE) based on retrieved oocytes has not been evaluated extensively. For the individualization of councelling for PGD it is crutial to know the availability of transfer and inform the patient accordingly. In this study we aim to determine the optimal number of oocytes to find at least one transferrable euploid embryo in PGD cases for aneuploidy testing, in addition to determine POFEE based on retrieved oocytes.

Live birth after PGD with confirmation by a comprehensive approach (karyomapping) for simultaneous detection of monogenic and chromosomal disorders

Preimplantation genetic diagnosis (PGD) for monogenic disorders has the drawback of time and cost associated with tailoring a specific test for each couple, disorder, or both. The inability of any single assay to detect the monogenic disorder in question and simultaneously the chromosomal complement of the embryo also limits its application as separate tests may need to be carried out on the amplified material. The first clinical use of a novel approach (‘karyomapping’) was designed to circumvent this problem. In this example, karyomapping was used to confirm the results of an existing PGD case detecting both chromosomal abnormalities and a monogenic disorder (Smith–Lemli–Opitz [SLO] syndrome) simultaneously. The family underwent IVF, ICSI and PGD, and both polar body and cleavage stage biopsy were carried out. Following whole genome amplification, array comparative genomic hybridisation of the polar bodies and minisequencing and STR analysis of single blastomeres were used to diagnose maternal aneuploidies and SLO status, respectively. This was confirmed, by karyomapping. Unlike standard PGD, karyomapping required no a-priori test development. A singleton pregnancy and live birth, unaffected with SLO syndrome and with no chromosome abnormality, ensued. Karyomapping is potentially capable of detecting a wide spectrum of monogenic and chromosome disorders and, in this context, can be considered a comprehensive approach to PGD.

Advances in preimplantation genetic diagnosis/screening.

Preimplantation genetic diagnosis (PGD) gives couples who have a high risk of transmitting genetic disorders to their baby the chance to have a healthy offspring through embryo genetic analysis and selection. Preimplantation genetic screening (PGS) is an effective method to select euploid embryos that may prevent repeated implantation failure or miscarriage. However, how and to whom PGS should be provided is a controversial topic. The first successful case of PGD of a human being was reported in 1990, and there have been tremendous improvements in this technology since then. Both embryo biopsy and genetic technologies have been improved dramatically, which increase the accuracy and expand the indications of PGD/PGS.

Preimplantation Genetic Diagnosis (PGD) for Genetic Prion Disorder Due to F198S Mutation in thePRNPGene

To describe the first case of preimplantation genetic diagnosis (PGD) and in vitro fertilization (IVF) performed for the prevention of genetic prion disease in the children of a 27-year-old asymptomatic woman with a family history of Gerstmann-Sträussler-Sheinker syndrome (GSS). PGD and fertilization cycles resulted in detection of 6 F198S mutation-free embryos. Of these, 2 were selected for embryo transfer to the patient's uterus, yielding a clinical twin pregnancy and birth of healthy but slightly premature offspring with normal development at age 27 months. IVF with PGD is a viable option for couples who wish to avoid passing the disease to their offspring. Neurologists should be aware of PGD to be able to better consult at-risk families on their reproductive choices.

Number of embryos to be biopsied is a strong predictive indicator for a successful preimplantation genetic diagnosis outcome in translocation cases

The objective of this study was to analyse if the pregnancy rate and cycle outcome of translocation carriers affected by the number of embryos biopsied for preimplantation genetic diagnosis (PGD)? Transfer and pregnancy rates of patients undergoing PGD for both Robertsonian and reciprocal translocations were retrospectively analysed from August 2010 to December 2012 in Bahceci Assisted Reproductive Technology Centres. Fifty cycles of PGD was performed for 45 couples carrying Robertsonian and reciprocal translocations by multicolour fluorescent in situ hybridisation (FISH) on day 3 post fertilisation. The genetic as well as cycle outcomes of PGD cases were examined and the successful pregnancies were followed up. The chance of finding a balanced embryo is considerably higher when nine or more embryos (group 1, 408 embryos) were biopsied and analysed in PGD cycles. When eight or less biopsied embryos were analysed (group 2, 123 embryos), the chance of finding a transferable embryo after PGD was 32% compared to77% in group 1. Therefore, the cancellation rate in group 2 is significantly higher than group 1. Furthermore, once a suitable embryo is found, the pregnancy rates were slightly higher in group 1 (38%) compared to the group 2 (33%) per embryo transfer. This study shows that the number of embryos analysed for translocations in PGD plays an important role in finding a balanced embryo and successful cycle outcome. If there are less than nine embryos biopsied for PGD, the likelihood of finding a balanced embryo is considerably reduced and the cycle cancellation risk is high. In these patients, performing multiple cycles of in vitro fertilisation and pooling the embryos by current cryopreservation techniques before PGD analysis can be advised.

Two different microarray technologies for preimplantation genetic diagnosis (PGD) and screening (PGS), due to reciprocal translocation imbalances, demonstrate equivalent clinical pregnancy rates

of chromosomal aneuploidy and single gene disorders (SGD) via PGD. This was done via multiplex PCR combined with array CGH (aCGH). DESIGN: Clinical research study. MATERIALS AND METHODS: A total of 47 IVF cycles were included in this study involving 44 couples and 25 different single gene disorders (HLA matching, autosomal dominant, recessive and X-linked), with mean maternal age of 32.9. Patient specific PCR protocols were optimized for simultaneous detection of mutations and polymorphic sites. PGD was performed by blastocyst biopsy, modified whole genome amplification, followed by analysis with aCGH for aneuploidy and by PCR for the SGD. Only euploid and unaffected/carrier embryos were considered for transfer. To confirm the diagnosis, 25 abnormal embryos were re-biopsied and reanalyzed using separate aCGH/molecular tests. RESULTS: From the 329 blastocyst samples tested in this clinical study, full results were obtained for both SGD and aCGH for 302 embryos (91.8%). Only 2 samples gave no results for both SGD and aCGH (0.61%). For the SGD 324/329 (98.5%) samples gave results, whereas 305/329 (92.7%) samples gave aCGH results. Amongst the embryos with full results, 57 (18.9%) were either normal or carriers of a SGD and euploid. If only SGD results were considered, 150 (49.7%) of the samples tested would have been suitable for transfer, but 93 (62%) would have been in fact aneuploid. Reanalysis of aCGH and SGD revealed a 0% error rate. Preliminary data shows improved implantation compared to PGD cases without aCGH. Among patients who previously had SGD PGD only, several have miscarried with aneuploid fetuses. CONCLUSION: The results show high rates of chromosome abnormalities among embryos that otherwise are unaffected by the SGD. This highlights the limitations of replacing embryos based solely on SGD PGD. The process of PGD is long and traumatic for patients. This may allow them to minimize the risk of miscarriage due to chromosome abnormalities.