RESEARCH QUESTION:What are the incidence and patterns of meiotic trisomies and recombination separately and in relation to each other at the blastocyst stage via single nucleotide polymorphism genotyping combined with array comparative genomic hybridization.DESIGN:Single nucleotide polymorphism microarrays were carried out on a total of 1442 blastocyst stage embryos derived from 268 fertile couples undergoing preimplantation genetic diagnosis for the purposes of avoiding transmittance of known single gene disorders to their offspring; 24-chromosome aneuploidy screening via array comparative genomic hybridization was carried out in parallel.RESULTS:One hundred per cent of meiotic trisomies identified in these embryos were of maternal origin and their incidence increased significantly with advancing maternal age (P < 0.0001). A total of 55.8% of meiotic trisomies were meiosis I-type and 44.2% were meiosis II-type. Certain chromosomes were affected more by meiosis I-type errors, whereas others experienced more meiosis II-type errors. A detailed recombination analysis was carried out for 11,476 chromosomes and 17,763 recombination events were recorded. The average number of recombination sites was 24.0 ± 0.3 for male meiosis and 41.2 ± 0.6 for female meiosis (autosomes only). Sex-specific differences were observed in the locations of recombination sites. Comparative analysis conducted between 190 euploid embryos and 69 embryos presenting maternal meiotic trisomies showed similar recombination rates (P = 0.425) and non-recombinant chromatid rates (P = 0.435) between the two categories; differences, however, were observed when analysing embryos affected with specific maternal meiotic trisomies.CONCLUSIONS:This study yielded unique data concerning recombination and the origin of aneuploidies observed during the first few days of life and provides a novel insight into these important biological processes.

PGD for BRCA testing offers a viable option for those women hoping to decrease disease in and increase survival of their children. We set out to characterize the outcomes of patients undergoing PGD to test for BRCA mutations along with PGS for aneuploidy assessment, and to determine the odds of delivery per retrieval. Retrospective Descriptive Series. All PGD cycles for the BRCA mutation (both BRCA1 and BRCA2) performed at a reference genetics laboratory were queried. Only cycles in which both PGS and PGD were performed were included. All methodologies of PGD and PGS were included in the analysis. Determination of delivery rate was based on an established live birth rate of 65% per euploidic embryo. 48 cycles resulted from the query and 42 cycles were included in the analysis. Twenty nine cycles were PGD for BRCA1 and 13 for BRCA2. The average maternal age was 33.29±4.27 (range 23-40) years with 6.76±4.29 (range 1-17) embryos for biopsy. The overall percentage of affected embryos for BRCA mutations was 52.36%±20.37% of which 40.11% ± 36.51% were found to be aneuploid. No unaffected euploid embryos were available for transfer in 26.19% (n=11) cases. When compared by age, no statistical significance was found in the number of embryos for biopsy, percent affected for BRCA mutation, the number of transferable (unaffected and euploid) embryos available, or chance of live birth following a single embryo transfer per cycle. The rate of aneuploid embryos in BRCA patients appears consistent with previously published rates of age associated aneuploidy(1). BRCA 1 and BRCA2 patients were independently analyzed, showing no statistical difference in any parameters evaluated. Combining PGD for BRCA and PGS for aneuploidy reduces the number of embryos available for transfer but allows for more specific selection of normals. Due to the rate of PGS/PGD normal being 14-29% of embryos, some women may be faced with considering the transfer of an effected male embryo. Patients undergoing embryo selection for BRCA should be well-informed of the odds of finding a PGS/PGD normal embryo, having no embryo to transfer, a male normal PGS carrier and pregnancy.Tabled 1PGD/PGS Results for BRCA Mutations Stratified by Age GroupageBRCA pos (%)Aneuploid Embryos (%)Aneuploid-BRCA pos Embryos (%)Female BRCA pos embryos (%)Male BRCApos (Embryos (%)Embryos for transfer (%)Chance of pregnancy per retrieval + single embryo transfer (%)24-30(n=10)51.51±21.7134.99±20.8455.0±25.355.00±25.3528.7±34.529.08±20.0418.90±13.0331-35(n=21)61.57±22.2938.73±27.8648.2±37.148.17±37.1356.8±35.224.69±22.4616.05±14.60>35 (n=11)54.05±19.5849.0±33.549.0±33.549.03±33.5444.6±33.313.75±13.388.94±8.70p0.4140.0690.8660.8690.1160.2250.225 Open table in a new tab

BackgroundRecent randomized trials have indicated the benefit of using comprehensive chromosome screening (CCS) methodologies and their potential of enhancing in vitro fertilization (IVF) success rates. Most recently a new methodology, known as next generation sequencing (NGS), was introduced into preimplantation genetic diagnosis (PGD) for CCS. NGS has been shown to be advantageous over other CCS methodologies, potentially offering higher precision of diagnosis [e.g. detection of abnormalities present only in some cells of the trophectoderm biopsied (mosaicism); a feature that is not available using other CCS techniques].ObjectiveTo clinically apply a methodology that combines PGD for single gene disorders with CCS-NGS of blastocysts through utilization of a single trophectoderm biopsy.Materials and methodsA total of 52 embryos derived from 11 different patients were assessed in this study. A single trophectoderm biopsy was carried out on day 5/6 of preimplantation development. Multiple displacement amplification was performed on the samples and aliquots from each amplified product were used to perform Karyomapping (Illumina, USA) for PGD of single gene disorders and NGS for CCS. The VeriSeq PGS assay and a MiSeq desktop sequencer (Illumina) were used for NGS. The BlueFuse Multi analysis software (Illumina) was utilized for interpretation of results.Results60% of the embryos were found to be either unaffected or carriers of the disorder being tested while, 40% of the embryos assessed were found to be euploid via NGS. 31.1% of the overall embryos assessed were seen to have at least one mosaic abnormality, while 8.9% of the embryos were determined to have only a single mosaic abnormality. Furthermore, segmental abnormalities were detected in 7 of the embryos tested and ranged from 7.8 to 104.6 megabases in size. A total of 16 embryos were found to be at low risk for the single gene disorder assessed and aneuploidy and could therefore be considered for transfer.ConclusionsThis study documents the successful clinical application of PGD for single gene disorders in combination with CCS-NGS using a single trophectoderm biopsy. The combination of powerful and highly accurate methodologies such as Karyomapping for single gene disorders and NGS for CCS, is expected to benefit patients undergoing IVF/PGD and can enhance their chances for a successful cycle leading to a healthy pregnancy.Financial supportInstitutional support.ReferencesNon applicable. BackgroundRecent randomized trials have indicated the benefit of using comprehensive chromosome screening (CCS) methodologies and their potential of enhancing in vitro fertilization (IVF) success rates. Most recently a new methodology, known as next generation sequencing (NGS), was introduced into preimplantation genetic diagnosis (PGD) for CCS. NGS has been shown to be advantageous over other CCS methodologies, potentially offering higher precision of diagnosis [e.g. detection of abnormalities present only in some cells of the trophectoderm biopsied (mosaicism); a feature that is not available using other CCS techniques]. Recent randomized trials have indicated the benefit of using comprehensive chromosome screening (CCS) methodologies and their potential of enhancing in vitro fertilization (IVF) success rates. Most recently a new methodology, known as next generation sequencing (NGS), was introduced into preimplantation genetic diagnosis (PGD) for CCS. NGS has been shown to be advantageous over other CCS methodologies, potentially offering higher precision of diagnosis [e.g. detection of abnormalities present only in some cells of the trophectoderm biopsied (mosaicism); a feature that is not available using other CCS techniques]. ObjectiveTo clinically apply a methodology that combines PGD for single gene disorders with CCS-NGS of blastocysts through utilization of a single trophectoderm biopsy. To clinically apply a methodology that combines PGD for single gene disorders with CCS-NGS of blastocysts through utilization of a single trophectoderm biopsy. Materials and methodsA total of 52 embryos derived from 11 different patients were assessed in this study. A single trophectoderm biopsy was carried out on day 5/6 of preimplantation development. Multiple displacement amplification was performed on the samples and aliquots from each amplified product were used to perform Karyomapping (Illumina, USA) for PGD of single gene disorders and NGS for CCS. The VeriSeq PGS assay and a MiSeq desktop sequencer (Illumina) were used for NGS. The BlueFuse Multi analysis software (Illumina) was utilized for interpretation of results. A total of 52 embryos derived from 11 different patients were assessed in this study. A single trophectoderm biopsy was carried out on day 5/6 of preimplantation development. Multiple displacement amplification was performed on the samples and aliquots from each amplified product were used to perform Karyomapping (Illumina, USA) for PGD of single gene disorders and NGS for CCS. The VeriSeq PGS assay and a MiSeq desktop sequencer (Illumina) were used for NGS. The BlueFuse Multi analysis software (Illumina) was utilized for interpretation of results. Results60% of the embryos were found to be either unaffected or carriers of the disorder being tested while, 40% of the embryos assessed were found to be euploid via NGS. 31.1% of the overall embryos assessed were seen to have at least one mosaic abnormality, while 8.9% of the embryos were determined to have only a single mosaic abnormality. Furthermore, segmental abnormalities were detected in 7 of the embryos tested and ranged from 7.8 to 104.6 megabases in size. A total of 16 embryos were found to be at low risk for the single gene disorder assessed and aneuploidy and could therefore be considered for transfer. 60% of the embryos were found to be either unaffected or carriers of the disorder being tested while, 40% of the embryos assessed were found to be euploid via NGS. 31.1% of the overall embryos assessed were seen to have at least one mosaic abnormality, while 8.9% of the embryos were determined to have only a single mosaic abnormality. Furthermore, segmental abnormalities were detected in 7 of the embryos tested and ranged from 7.8 to 104.6 megabases in size. A total of 16 embryos were found to be at low risk for the single gene disorder assessed and aneuploidy and could therefore be considered for transfer. ConclusionsThis study documents the successful clinical application of PGD for single gene disorders in combination with CCS-NGS using a single trophectoderm biopsy. The combination of powerful and highly accurate methodologies such as Karyomapping for single gene disorders and NGS for CCS, is expected to benefit patients undergoing IVF/PGD and can enhance their chances for a successful cycle leading to a healthy pregnancy. This study documents the successful clinical application of PGD for single gene disorders in combination with CCS-NGS using a single trophectoderm biopsy. The combination of powerful and highly accurate methodologies such as Karyomapping for single gene disorders and NGS for CCS, is expected to benefit patients undergoing IVF/PGD and can enhance their chances for a successful cycle leading to a healthy pregnancy.

Karyomapping (Kmap) is a linkage-based single nucleotide polymorphism technology proven to be highly efficient in PGD diagnosis and applicable to most single gene disorders (SGD). Here, outcomes of such testing are reviewed. Kmap with and without comprehensive chromosome screening (CCS) via array comparative genomic hybridization (aCGH) (24sure, Illumina) or next generation sequencing (NGS) (VeriSeq, Illumina) was used to screen embryos (blastocysts) undergoing PGD for SGDs. Between 1/2014-4/2016, PGD was performed on 694 cycles (4284 blastocyst biopsies) with subsequent cryopreservation. Each sample was whole genome amplified and analyzed using Kmap (Illumina, USA). Additionally, 96.1% (609/694) of cases had CCS. Follow-up data was obtained for 478 cycles with embryos suitable for transfer (free from SGD and euploid if CCS performed). At the time of data collection, 223 cycles had undergone embryo transfer. The 694 Kmap cycles comprised 105 SGDs, human leukocyte antigen (HLA) matching, and microdeletions/duplications. Diagnostic results were available for 97% (4154/4284) of samples. Implantation rate was 75.9% (151/199) for PGD+CCS (average maternal age (MA): 32.9 years) and 64% (16/25) for PGD only (average MA: 34.3 years). The pregnancy rate per transfer was 74.7% (124/166) for PGD+CCS and 78.9% (15/19) for PGD only. Single embryo transfer was performed for most; 20 had double embryo transfer. The average number of embryos suitable for transfer per cycle was 1.95 (PGD+CCS) and 2.56 (PGD only). A total of 30 live births and 96 ongoing pregnancies were reported. Confirmatory testing via chorionic villus sampling (CVS) or amniocentesis was performed for 6 pregnancies. Follow-up testing via newborn screening panel for 9 live births was in concordance with Kmap diagnosis. Results were in complete concordance with Kmap diagnosis; no misdiagnoses have been reported to date from the 121 cycles with successful pregnancy outcomes. With the increase in patient awareness regarding availability of PGD and the rise of preconception carrier screening, there is a growing demand for PGD. Higher implantation rates were observed for patients who underwent CCS in combination with Kmap. Pregnancy rates were similar as sample size for “PGD only” was smaller and 62.5% of “PGD only” patients had a double embryo transfer. Due to the high diagnostic accuracy, comprehensive analysis and short preparation time, Kmap is a successful treatment strategy for patients requesting PGD for inherited disorders.

Preimplantation Genetic Diagnosis (PGD) has helped couples avoid inherited genetic diseases for over 25 years. Karyomapping can streamline this process with a high resolution and rapid result test design. This study is an evaluation of the Single Nucleotide Polymorphism (SNP)-based karyomapping platform as the best standard for preimplantation genetic diagnosis of single gene disorders, using an analysis of over 9,000 trophectoderm biopsies and 304 diverse genetic conditions. Retrospective, multi-faceted analysis of embryo biopsies submitted for single-gene diagnosis on the new karyomapping test platform. Familial DNA from either buccal swabs or blood samples was analyzed along with amplified embryo DNA using the Illumina Karyomapping assay. The data was analyzed with BlueFuse Multi software to detect affected familial haplotypes and determine disease status of embryos. When requested, aneuploidy screening was performed in parallel using either Comparative Genomic Hybridization microarray (aCGH) or Next Generation Sequencing. 1,417 families were analyzed using the karyomapping technology, with an average PGD probe design length of 4 weeks. In total, 9,426 blastocyst biopsies were assessed, averaging 6.8 samples per couple. Of the samples tested, 4.8% were given a "no result" diagnosis because quality control metrics were not met, or the sample had poor amplification. 1.4% were given a "no diagnosis" or "inconclusive" result due to sample contamination or partial/incomplete results. The remaining 8,848 biopsies (93.9%) were successfully diagnosed for 304 different genetic conditions. Karyomapping alone was performed on 294 cases (2,013 samples), resulting in a transfer rate (proportion of assessed embryos available for transfer) of 46.7%. Aneuploidy screening was included in the remaining 1,123 cases, resulting in a transfer rate of 29.9%. Karyomapping has proven to be a reliable and reproducible test, as evidenced by the robust diagnosis of 93.9% of the 9,426 biopsies analyzed over the course of 2 years. With karyomapping, the vast number of data points available across the entire genome allows for diagnosis of common, uncommon, and novel genetic disorders with a single standard test platform. This is in stark contrast to the time-consuming customization required by the previous methodology using short tandem repeats. The families undergoing PGD are benefiting from this technology, particularly those in need of a rapid test design. Great success with this technology demonstrates that karyomapping has become the new gold standard for PGD.

To document and report the relevance and implications of incidental findings identified during PGD in an effort to bring awareness and emphasize the need for implementing a standard operating protocol to report such findings to patients and their physicians. Incidental findings identified in couples undergoing PGD for gene disorders were recorded and reported from July 2015 to March 2016. During the 9 month period, 269 cases were prepared through utilization of single nucleotide polymorphism (SNP) arrays (Karyomapping; Illumina, USA). DNA samples were obtained from couples and family members for test preparation purposes. During evaluation of sample quality, review of the SNP array profiles was completed. All incidental findings were reported to the physician and patient and recommendation for follow-up microarray testing was provided. Incidental findings were identified in 10/269 PGD test preparations, affecting chromosomes 4, 14, 15, 16, 22, and X. Nine were microduplications and 1 was a microdeletion. The size of microduplications detected ranged from 0.18 megabases (Mb) to 3.5 Mb. Interestingly, three of the microduplications detected (4q35.2, 15q11.2, and 16q23.3) were each seen in 2 separate cases, accounting for 6/10 cases. The microdeletion identified (located on chromosome X) was determined to be of considerable size at 28Mb, potentially associated with health implications in the carrier female. Follow-up microarray analysis was pursued in 7 of the cases, as 3 of the patients declined further evaluation. Three of the incidental findings were reported as variants of uncertain significance (VUS), and 3 were reported as normal population variants. Results are still pending on 1 case. One patient elected to pursue PGD for the microduplication. Results from 2 PGD cycles for this patient with a total of 9 embryos, revealed 3 embryos free of the microduplication, but a total of 1 embryo available for transfer as the other 2 were affected with the single gene disorder and/or aneuploidy. With the implementation of new and advanced methodologies in clinical practice, it is becoming progressively more common to incidentally obtain information that is additional to the requested test but may have significant health implications to the patient, other family members and/or future children. It is vital to acknowledge and address this issue in the clinical laboratory setting and a standard operating procedure has to be in place to handle such situations.

Primary ovarian insufficiency associated with FXS makes it challenging to retrieve multiple oocytes necessary for PGD. Moreover, the limited amount of embryonic DNA material available presents some challenges for a comprehensive test. PGD for FXS with aCGH allows embryo selection by detecting CGG repeat sizes associated with FXS (distinction of normal, intermediate or premutation from full mutation allele range) combined with chromosomal copy screening (CCS) following in-vitro fertilization. To evaluate results for the simultaneous analysis of CCS via aCGH and FXSPGD with direct repeat size determination via PCR and to assess aneuploidy rates among patients undergoing PGD for FXS. Overall amplification and diagnosis results for FXS direct mutation testing combine with aCGH were evaluated from 223 blastocysts consisting of 41 PGD cycles, involving two age groups (<35years and 35-44years). From the 41 PGD cycles, 26 cycles (165 blastocysts) were from patients <35y (mean 31.4+2.4y), and 12 cycles (58 blastocysts) were from patients 35-44y (mean 40.1 + 3.2y). Complete diagnostic results (FXS and aCGH) were obtained on 95.8% (158/165) for the <35y and 87.9% (51/58) for 35-44y. Average euploidy rate for the <35 age group was 59.5% (94/158) whereas euploidy rate for the 35-44y group was 29.4.X% (15/51). Amongst the embryos with euploidy results, FXS repeat allele size assessment indicated 68.1% (64/94) and 93.3 % (14/15) were free from FXS full mutation for the <35y and 35-44y groups, respectively. Based on the FXS and aCGH results, all 26 cycles from the <35y group had at least one embryo suitable for transfer compared to only 6 /12 cycles from the 35-44y group. The results show high rates of chromosome abnormalities among both age groups and, as expected, considerably higher rates in the 35-44y group. These rates agree with previous published aneuploidy rates per maternal age from non-FX patients [1]. This highlights the importance to select a comprehensive diagnosis strategy, FXS with direct mutation and CCS, regardless of the maternal age group in order to enhance embryo selection, which may lead to a short time to pregnancy. This is especially important for patients with poor ovarian reserve.

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.

Currently, the methods available for preimplantation genetic diagnosis (PGD) of in vitro fertilized (IVF) embryos do not detect de novo single-nucleotide and short indel mutations, which have been shown to cause a large fraction of genetic diseases. Detection of all these types of mutations requires whole-genome sequencing (WGS). In this study, advanced massively parallel WGS was performed on three 5- to 10-cell biopsies from two blastocyst-stage embryos. Both parents and paternal grandparents were also analyzed to allow for accurate measurements of false-positive and false-negative error rates. Overall, >95% of each genome was called. In the embryos, experimentally derived haplotypes and barcoded read data were used to detect and phase up to 82% of de novo single base mutations with a false-positive rate of about one error per Gb, resulting in fewer than 10 such errors per embryo. This represents a ∼ 100-fold lower error rate than previously published from 10 cells, and it is the first demonstration that advanced WGS can be used to accurately identify these de novo mutations in spite of the thousands of false-positive errors introduced by the extensive DNA amplification required for deep sequencing. Using haplotype information, we also demonstrate how small de novo deletions could be detected. These results suggest that phased WGS using barcoded DNA could be used in the future as part of the PGD process to maximize comprehensiveness in detecting disease-causing mutations and to reduce the incidence of genetic diseases.

Assessment of a universal SNP array based protocol, known as Karyomapping, as a PGD treatment strategy for patients requesting embryo testing for inherited disorders. Karyomapping utilizes a universal protocol, applicable to virtually all patients, for linkage-based PGD. It eliminates the need to develop and optimize patient-specific protocols, drastically reducing the waiting time for initiation of IVF treatment. This technology was previously validated using the gold-standard polymerase chain reaction methodology (with 100% concordance) and has proven to be highly efficient for even the most complex of cases, including those not feasible using conventional PGD methods.Among the 300 Karyomapping cases performed between December 2013 and May 2015, clinical outcome and follow-up data was collected for 223 PGD cycles with transferable embryos. Patients were referred from over 50 IVF centers in the USA. Embryos were biopsied at the blastocyst stage and frozen for transfer in subsequent cycle(s). Samples were whole genome amplified and analyzed using Karyomapping combined with direct mutation detection as needed and/or independent comprehensive chromosome screening (CCS) when requested (84% cases). The diagnostic rates were 98.7% for Karyomapping and 98.6% for CCS. The average numbers of embryos analyzed and eligible for transfer per cycle were 5.3 and 2.5 for PGD alone, and 6.2 and 2.3 for PGD+CCS. Batching of embryos prior to analysis was carried out for 11% of PGD+CCS cases (2% for PGD alone).The CCS improved clinical outcome. The implantation and clinical pregnancy rates were 70.5% (43/61) and 64% (34/53) for PGD+CCS cycles, versus 54.5% (6/11) and 55.6% (5/9) for PGD alone. Most transfers involved a single thawed blastocyst, except for ten patients (16%) who had two embryos transferred at once. The average maternal ages were 32.3 and 34.2 years for PGD and PGD+CCS, respectively. There are 13 live births and 36 ongoing pregnancies to date. Prenatal and perinatal testing was performed in four cases confirming PGD in all instances. The high clinical pregnancy rates were similar to outcomes achieved by the most successful in-vitro fertilization (IVF) programs. As lengthy patient-specific test developments were unnecessary, greater numbers of patients were treated with faster time to diagnosis, transfer and pregnancy. SNP arrays thus allow PGD laboratories to deal with the rapidly growing demand for embryo testing for inherited disorders. The application of Karyomapping with simultaneous CCS speeds up the testing process and further improves clinical outcomes.

To evaluate the utilization of a comprehensive diagnosis strategy for preimplantation genetics diagnosis (PGD) of fragile X syndrome (FXS) in order to enhance selection of embryos suitable for transfer and increase therefore, the chances of patients achieving a pregnancy. Trophectoderm biopsies obtained from blastocysts preimplantation embryos were processed for direct FXS repeat size determination and linkage analysis via Karyomapping (Illumina, USA). When requested, comprehensive chromosome screening (CCS) via array comparative genomic hybridization testing was also performed in parallel to PGD for FXS. Trophectoderm biopsies were sent from 27 different IVF clinics between December 2013 and March 2015 to a single PGD laboratory for analysis. A total 168 blastocysts from 34 PGD cycles (4.9 embryos per cycle) were assessed for FXS direct mutation test (average maternal age: 34.8). Diagnostic results were obtained for 96.4% of the samples (162/168). The proportion of embryos found to be available for transfer based on the inheritance of the normal allele via linkage analysis was 53.7% (87/162). Among the 46.3% (75/162) categorized as having inherited the affected allele, direct triplet repeat sizing allowed the identification of 24.1% (39/162) additional blastocysts suitable for transfer (carriers of premutation or intermediate expansions) (p<0.001). Out of these cases, 26 cycles totaling 142 blastocysts were also tested for CCS in paralleled with FXS. Despite of the added CCS, 40.1% (57/142) of blastocysts were considered for transfer. Preliminary clinical outcome data showed a high pregnancy rate per transfer of 72.7% (8/11). There is significant diagnostic advantage on performing triplet repeat sizing to differentiate between embryos having inherited the permutation/intermediate from the full mutation triplet repeat expansion. Determination of the actual repeat expansion results in a significant increase in the number of blastocysts available for transfer. The strategy followed in this study resulted in high pregnancy rates, despite the potentially fewer embryos available for testing at the blastocyst stage. It is advisable to opt for a PGD strategy with more comprehensive diagnosis in order to enhance embryonic selection and increase therefore, the chances of achieving a pregnancy; something which is especially important in cases hindered by poor ovarian response.

Purpose: Technological advances now allow for multiplex platforms to simultaneously test many genetic conditions. Typically, such platforms are validated by assaying samples with known genotypes and/or phenotypes and/or with synthetic plasmids; however, these methods have limitations and with the inclusion of rarer diseases and mutations, we can no longer rely solely on them. We used a novel genomic database to validate an expanded genetic carrier screening platform. Methods: Our expanded carrier screening assay uses the Illumina Infinium iSelect HD Custom genotyping platform to test for 213 genetic diseases by assaying 1,663 pathogenic mutations. We leveraged two Coriell Institute biorepositories for validation: the Subcollection of Heritable Diseases and the 1000 Genomes Project. Results: We measured 12,394 mutation observations in 206 samples, resulting in 246 true positives, 12,147 true negatives, 1 false positive, and no false negatives. Results demonstrated high sensitivity (99.99%) and specificity (99.99%). Conclusion: We successfully validated our platform with two biorepositories, demonstrating high sensitivity and specificity. The 1000 Genomes Project samples provided both positive and negative validation for mutations in genes not available through other biorepositories, expanding the depth of validated variants. We recommend including samples from the 1000 Genomes Project in the validation of future multiplex testing platforms. Genet Med advance online publication 30 July 2015