Genetic diseases and aneuploidies can be detected with a single blastocyst biopsy: a successful clinical approach.

STUDY QUESTION: Can simultaneous detection of aneuploidies and genetic diseases or chromosomal aberrations in blastocysts reduce the chance of transferring embryos with low implantation potential, guaranteeing good clinical outcomes? SUMMARY ANSWER: The screening for chromosomal aneuploidies revealed that 50.6% of blastocysts diagnosed free of genetic disease or balanced, were aneuploid, therefore avoiding the transfer of blastocysts potentially resulting in implantation failures, miscarriages, or in some cases, in health affected live births. WHAT IS KNOWN ALREADY: PGD is applied in patients at risk of transmitting genetically inheritable diseases to their offspring. It has been demonstrated that aneuploidies can involve chromosomes other than those investigated with PGD, affecting embryo implantation competence. Performing the biopsy at blastocyst level produces higher clinical outcomes allowing a more accurate diagnosis, compared to blastomere biopsy. STUDY DESIGN, SIZE, DURATION: This consecutive case series study was performed from October 2011 to May 2016. Clinical and biological outcomes from 1122 blastocysts obtained in 304 PGD cycles for monogenic diseases (N = 163) or chromosomal rearrangements (N = 141) were analyzed. When the blastocyst resulted transferable after the PGD analysis or chromosomal rearrangement analysis, its ploidy status by mean of preimplantation genetic screening (PGS) was also detected using the same biopsy sample. Mean female age was 35.4 ± 4.2 years old. All biopsies were performed at blastocyst stage and analyzed by Whole Genome Amplification (WGA) followed by PCR for monogenic diseases, and by array-comparative genotype hybridization (array-CGH) for all cycles. PARTICIPANTS/MATERIALS, SETTING, METHOD: All mature oocytes retrieved were injected and cultured individually until the blastocyst stage at 37°C, 6% CO2, 5% O2. When the blastocyst was formed, it was biopsied and vitrified, awaiting the genetic results. The frozen-thawed embryo transfer was performed in a subsequent cycle. In some cases, when the blastocyst was obtained within the morning of Day 5 of culture, it had been maintained in culture and transferred on Day 6, after receiving the genetic report. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 2809 (2718 fresh and 91 frozen-thawed) mature oocytes were injected with a fertilization rate of 75.5% (N = 2120), leading to the development of 2102 embryos. A further 24 frozen embryos, previously vitrified without any genetic testing, were successfully warmed for genetic screening. A total of 2126 embryos were cultured with a blastocyst formation rate of 52.8% (N = 1122); all of them were biopsied from Day 4 to Day 7 of culture. After the genetic analysis, 309 (27.5%) blastocysts resulted transferable, both for monogenic disease or translocation and for their ploidy status, 42 were diploid/aneuploid mosaic, 55 were no result and 716 were not transferable, due to genetic disease or chromosomal rearrangement and/or for their ploidy status. Of note, 316 (50.6% of transferable blastocysts after PGD and 28.2% of total number of biopsied blastocysts) of the blastocysts resulted healthy for the genetic disease or chromosomal rearrangement were aneuploid. Out of 304 PGD/PGS cycles performed, 28.6% (N = 87) resulted in no-transferable blastocysts after only PGD analysis; this percentage increased to 39.8% (N = 121) when also PGS was carried out (Mc Nemar test P < 0.001). A total of 202 embryo-transfers were performed, 53 fresh and 149 cryopreserved, in which 218 healthy or carrier euploid blastocysts were transferred. Clinical pregnancy, implantation and miscarriage rates were 49.0, 47.7 and 9.9%, respectively. To date, 66 deliveries occurred with 70 healthy babies born and 13 pregnancies are still ongoing. Finally, 91 euploid healthy blastocysts are still cryopreserved waiting to be transferred. LIMITATIONS, REASONS FOR CAUTION: A higher than expected cycle cancellation rate could be found due to the double genetic analysis performed. For this reason, particular care should be taken in drafting and explaining informed consent, in order to avoid patient drop out. WIDER IMPLICATIONS OF THE FINDINGS: When the biopsy has to be performed in order to prevent the transmission of an inheritable disease, it should be mandatory to analyze also the genetic status of the blastocyst, avoiding useless embryo-transfers in this particular category of patients. In our study, 316 aneuploid healthy blastocysts could have been transferred without performing PGS, leading to implantation failures, miscarriages, or in some cases, to live births affected by different syndromes. STUDY FUNDING/COMPETING INTEREST(S): No specific funding was obtained for this study. None of the authors have any competing interests to declare. TRIAL REGISTRATION NUMBER: Not applicable.

Application of next-generation sequencing technology for comprehensive aneuploidy screening of blastocysts in clinical preimplantation genetic screening cycles.

STUDY QUESTION: Can next-generation sequencing (NGS) techniques be used reliably for comprehensive aneuploidy screening of human embryos from patients undergoing IVF treatments, with the purpose of identifying and selecting chromosomally normal embryos for transfer? SUMMARY ANSWER: Extensive application of NGS in clinical preimplantation genetic screening (PGS) cycles demonstrates that this methodology is reliable, allowing identification and transfer of euploid embryos resulting in ongoing pregnancies. WHAT IS KNOWN ALREADY: The effectiveness of PGS is dependent upon the biology of the early embryo and the limitations of the technology. Fluorescence in situ hybridization, used to test for a few chromosomes, has largely been superseded by microarray techniques that test all 24 chromosomes. Array comparative genomic hybridization (array-CGH) has been demonstrated to be an accurate PGS method and has become the de facto gold standard, but new techniques, such as NGS, continue to emerge. STUDY DESIGN, SIZE, DURATION: The study consisted of a prospective trial involving a double blind parallel evaluation, with both NGS and array-CGH techniques, of 192 blastocysts obtained from 55 consecutive clinical PGS cycles undertaken during the period of September to October 2013. Consistency of NGS-based aneuploidy detection was assessed by matching the results obtained with array-CGH-based diagnoses. Primary outcome measure was accuracy of the chromosomal analysis; secondary outcome measures were clinical outcomes. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Fifty-five patients (median age 39.3 years, range 32-46) undergoing PGS were enrolled in the study. All embryos were cultured to blastocyst stage; trophectoderm biopsy was performed on Day 5 of development or Day 6/7 for slower growing embryos. The method involved whole genome amplification followed by both NGS and array-CGH. The MiSeq control software, real-time analysis and reporter performed on-board primary and secondary bioinformatics analysis. Copy number variation analysis was accomplished with BlueFuse Multi software. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 192 blastocysts were blindly evaluated with the NGS-based protocol. Paired comparison between NGS and array-CGH from individual embryos showed concordant results in 191/192 (99.5%) of the blastocysts tested. In total 4608 chromosomes were assessed, 211 (4.6%) of which carried a copy number imbalance. NGS specificity for aneuploidy calling (consistency of chromosome copy number assignment) was 99.98% (4333/4334; 95% confidence interval [95% CI]: 99.87-100) with a sensitivity of 100% (211/211, 95% CI: 99.25-100). Despite one discordant result, NGS specificity and sensitivity for aneuploid embryo calling (24-chromosome diagnosis consistency) were both 100% since the discordant sample presented several other aneuploidies. Clinical application of the NGS-based approach revealed 74/192 (38.5%) euploid blastocysts. Following transfer of 50 embryos in 47 women, 34 women had positive hCG levels: 30 pregnancies continued, confirmed by at least one fetal sac and heart beat (63.8% clinical pregnancy rate/embryo transfer), 3 were biochemical and 1 miscarried. A total of 32 embryos implanted and led to the presence of a fetal sac (64.0% implantation rate). All pregnancies went to term resulting in the birth of 31 healthy babies. LIMITATION, REASON FOR CAUTION: Although clinical results reported high pregnancy outcomes following transfer of screened embryos, further data and broad-based clinical application are required to better define the role of NGS in PGS. Before recommending widespread application, a randomized controlled trial confirming its clinical effectiveness is advisable. WIDER IMPLICATION OF THE FINDING: This is the first study reporting extensive application of NGS-based comprehensive aneuploidy screening on embryos at blastocyst stage in a clinical setting versus array-CGH as test of reference. NGS has demonstrated a reliable methodology, with the potential to improve chromosomal diagnosis on embryos especially in terms of high-throughput, automation and ability to detect aneuploidy. NGS methodology may represent a valuable alternative to the other comprehensive aneuploidy screening techniques currently available. STUDY FUNDING/COMPETING INTERESTS: No external funding was sought for this study. Drs F.K. and C.-E.M. are full-time employees of Illumina, Inc., which provided NGS library and sequencing reagents for the study. All other authors have no conflicts to declare. TRIAL REGISTRATION NUMBER: Not applicable.

A highly specific q-RT-PCR assay to address the relevance of the JAK2WT and JAK2V617F expression levels and control genes in Ph-negative myeloproliferative neoplasms.

In Ph- myeloproliferative neoplasms, the quantification of the JAK2V617F transcripts may provide some advantages over the DNA allele burden determination. We developed a q-RT-PCR to assess the JAK2WT and JAK2V617F mRNA expression in 105 cases (23 donors, 13 secondary polycythemia, 22 polycythemia vera (PV), 38 essential thrombocythemia (ET), and 9 primary myelofibrosis (PMF)). Compared with the standard allele-specific oligonucleotide (ASO)-PCR technique, our assay showed a 100 % concordance rate detecting the JAK2V617F mutation in 22/22 PV (100 %), 29/38 (76.3 %) ET, and 5/9 (55.5 %) PMF cases, respectively. The sensitivity of the assay was 0.01 %. Comparing DNA and RNA samples, we found that the JAK2V617F mutational ratios were significantly higher at the RNA level both in PV (p = 0.005) and ET (p = 0.001) samples. In PV patients, JAK2WT expression levels positively correlated with the platelets (PLTs) (p = 0.003) whereas a trend to negative correlation was observed with the Hb levels (p = 0.051). JAK2V617F-positive cases showed the lowest JAK2WT and ABL1 mRNA expression levels. In all the samples, the expression pattern of beta-glucoronidase (GUSB) was more homogeneous than that of ABL1 or ß2 microglobulin (B2M). Using GUSB as normalizator gene, a significant increase of the JAK2V617F mRNA levels was seen in two ET patients at time of progression to PV. In conclusion, the proposed q-RT-PCR is a sensitive and accurate method to quantify the JAK2 mutational status that can also show clinical correlations suggesting the impact of the residual amount of the JAK2WT allele on the Ph- MPN disease phenotype. Our observations also preclude the use of ABL1 as a housekeeping gene for these neoplasms.

Combined TP53 status in tumor-free resection margins and circulating microRNA profiling predicts the risk of locoregional recurrence in head and neck cancer.

Locoregional recurrences represent a frequently unexpected problem in head and neck squamous cell carcinoma (HNSCC). Relapse often (10-30%) occurs in patients with histologically negative resection margins (RMs), probably due to residual tumor cells or hidden pre-cancerous lesions in normal mucosa, both missed by histopathological examination. Therefore, definition of a 'clean' or tumor-negative RM is controversial, demanding for novel approaches to be accurately explored. Here, we evaluated next generation sequencing (NGS) and digital PCR (dPCR) as tools to profile TP53 mutational status and circulating microRNA expression aiming at scoring the locoregional risk of recurrence by means of molecular analyses. Serial monitoring of these biomarkers allowed identifying patients at high risk, laying the ground for accurate tracking of disease evolution and potential intensification of post-operative treatments. Additionally, our pipeline demonstrated its applicability into the clinical routine, being cost-effective and feasible in terms of patient sampling, holding promise to accurately (re)-stage RMs in the era of precision medicine.

NIPAT as Non-Invasive Prenatal Paternity Testing Using a Panel of 861 SNVs.

In 1997, it was discovered that maternal plasma contains Cell-Free Fetal DNA (cffDNA). cffDNA has been investigated as a source of DNA for non-invasive prenatal testing for fetal pathologies, as well as for non-invasive paternity testing. While the advent of Next Generation Sequencing (NGS) led to the routine use of Non-Invasive Prenatal Screening (NIPT or NIPS), few data are available regarding the reliability and reproducibility of Non-Invasive Prenatal Paternity Testing (NIPPT or NIPAT). Here, we present a non-invasive prenatal paternity test (NIPAT) analyzing 861 Single Nucleotide Variants (SNV) from cffDNA through NGS technology. The test, validated on more than 900 meiosis samples, generated log(CPI)(Combined Paternity Index) values for designated fathers ranging from +34 to +85, whereas log(CPI) values calculated for unrelated individuals were below -150. This study suggests that NIPAT can be used with high accuracy in real cases.

Cross-Validation of Next-Generation Sequencing Technologies for Diagnosis of Chromosomal Mosaicism and Segmental Aneuploidies in Preimplantation Embryos Model.

Detection of mosaic embryos is crucial to offer more possibilities of success to women undergoing in vitro fertilization (IVF) treatment. Next Generation Sequencing (NGS)-based preimplantation genetic testing are increasingly used for this purpose since their higher capability to detect chromosomal mosaicism in human embryos. In the recent years, new NGS systems were released, however their performance for chromosomal mosaicism are variable. We performed a cross-validation analysis of two different NGS platforms in order to assess the feasibility of these techniques and provide standard parameters for the detection of such aneuploidies. The study evaluated the performance of MiseqTM Veriseq (Illumina, San Diego, CA, USA) and Ion Torrent Personal Genome Machine PGMTM ReproSeq (Thermo Fisher, Waltham, MA, USA) for the detection of whole and segmental mosaic aneuploidies. Reconstructed samples with known percentage of mosaicism were analyzed with both platforms and sensitivity and specificity were determined. Both platforms had high level of specificity and sensitivity with a Limit Of Detection (LOD) at ≥30% of mosaicism and a showed a ≥5.0 Mb resolution for segmental abnormalities. Our findings demonstrated that NGS methodologies are capable of accurately detecting chromosomal mosaicism and segmental aneuploidies. The knowledge of LOD for each NGS platform has the potential to reduce false-negative and false-positive diagnoses when applied to detect chromosomal mosaicism in a clinical setting.

Cross-sectional analysis of circulating tumor DNA in primary colorectal cancer at surgery and during post-surgery follow-up by liquid biopsy.

BACKGROUND: Liquid biopsy (LB) in early-stage, non-metastatic colorectal cancer (CRC) must be sensitive enough to detect extremely low circulating tumor DNA (ctDNA) levels. This challenge has been seldom and non-systematically investigated. METHODS: Next generation sequencing (NGS) and digital PCR (dPCR) were combined to test tumor DNAs (tDNAs) and paired ctDNAs collected at surgery from 39 patients, 12 of whom were also monitored during the immediate post-surgery follow up. Patients treated for metastatic disease (n = 14) were included as controls. RESULTS: NGS and dPCR concordantly (100% agreement) called at least one single nucleotide variant (SNV) in 34 tDNAs, estimated differences in allelic frequencies being negligible (±1.4%). However, despite dPCR testing, SNVs were only detectable in 15/34 (44.1%) ctDNAs from patients at surgery, as opposed to 14/14 (100%) metastatic patients. This was likely due to striking differences (average 10 times, up to 500) in ctDNA levels between groups. NGS revealed blood-only SNVs, suggesting spatial heterogeneity since pre-surgery disease stages, and raising the combined NGS/dPCR sensitivity to 58.8%. ctDNA levels at surgery correlated with neither tumor size, stage, grade, or nodal status, nor with variant abundance in paired tDNA. LB sensitivity reached 63.6% when ctDNA was combined with CEA. Finally, persistence and absence of ctDNA on the first conventional (month 3) post-surgery follow-up were associated with fast relapse and a disease-free status in 3 and 7 patients, respectively. CONCLUSIONS: A simple clinical NGS/dPCR/CEA combination effectively addresses the LB challenge in a fraction of non-metastatic CRC patients.

Extent of chromosomal mosaicism influences the clinical outcome of in vitro fertilization treatments.

OBJECTIVE: To assess whether the extent of chromosomal mosaicism can influence the success rate of IVF treatments. DESIGN: Prospective study. SETTING: Private genetic and assisted reproduction centers. PATIENT(S): The transfer of mosaic embryos was offered to 77 women for which IVF resulted in no euploid embryos available for transfer. INTERVENTION(S): All embryos were cultured to blastocyst stage; trophectoderm biopsy was performed on day 5/6 of development. Comprehensive chromosome screening was performed using either next-generation sequencing or array-comparative genomic hybridization methodologies. MAIN OUTCOME MEASURE(S): The clinical outcome obtained after transfer of mosaic embryos with low (<50%) and high (≥50%) aneuploidy percentage was compared with that resulting from a control group of 251 euploid blastocysts. RESULT(S): A significantly higher implantation rate (48.9% vs. 24.2%), clinical pregnancy rate/ET (40.9% vs. 15.2%), and live-birth rate (42.2% vs. 15.2%) were observed comparing embryos with mosaicism <50% and ≥50%. Mosaic embryos with high aneuploidy percentage (≥50%) showed a significantly lower clinical pregnancy rate/ET (15.2% vs. 46.4%), implantation rate (24.4% vs. 54.6%), and live-birth rate (15.2% vs. 46.6%) than euploid blastocysts. In contrast, embryos with lower aneuploidy percentage (<50%) have a clinical outcome similar to euploid embryos. CONCLUSION(S): The results of this study further confirm that mosaic embryos can develop into healthy euploid newborns. We demonstrated that the extent of mosaicism influences the IVF success rate. Mosaic embryos with low aneuploidy percentage have higher chances of resulting in the birth of healthy babies compared with embryos with higher mosaicism levels.

Development and validation of a next-generation sequencing-based protocol for 24-chromosome aneuploidy screening of embryos.

OBJECTIVE: To validate a next-generation sequencing (NGS)-based method for 24-chromosome aneuploidy screening and to investigate its applicability to preimplantation genetic screening (PGS). DESIGN: Retrospective blinded study. SETTING: Reference laboratory. PATIENT(S): Karyotypically defined chromosomally abnormal single cells and whole-genome amplification (WGA) products, previously analyzed by array comparative genomic hybridization (array-CGH), selected from 68 clinical PGS cycles with embryos biopsied at cleavage stage. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Consistency of NGS-based diagnosis of aneuploidy compared with either conventional karyotyping of single cells or array-CGH diagnoses of single blastomeres. RESULT(S): Eighteen single cells and 190 WGA products from single blastomeres, were blindly evaluated with the NGS-based protocol. In total, 4,992 chromosomes were assessed, 402 of which carried a copy number imbalance. NGS specificity for aneuploidy call (consistency of chromosome copy number assignment) was 99.98% (95% confidence interval [CI] 99.88%-100%) with a sensitivity of 100% (95% CI 99.08%-100%). NGS specificity for aneuploid embryo call (24-chromosome diagnosis consistency) was 100% (95% CI 94.59%-100%) with a sensitivity of 100% (95% CI 97.39%-100%). CONCLUSION(S): This is the first study reporting extensive preclinical validation and accuracy assessment of NGS-based comprehensive aneuploidy screening on single cells. Given the high level of consistency with an established methodology, such as array-CGH, NGS has demonstrated a robust high-throughput methodology ready for clinical application in reproductive medicine, with potential advantages of reduced costs and enhanced precision.