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.

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.

The clinical utility of genome-wide non invasive prenatal screening.

OBJECTIVE: In this study, we expanded conventional cell-free fetal DNA (cfDNA)-based non-invasive prenatal testing (NIPT) to cover the entire genome. We aimed to compare the performance of the two tests in a large general population of pregnant women, in order to assess the clinical utility of the genome-wide screening. METHOD: Genome-wide cfDNA analysis was offered to 12 114 pregnant women undergoing NIPT for common fetal aneuploidy. Sequencing data were analyzed using an algorithm optimized to identify aneuploidies and subchromosomal aberrations. RESULTS: Genome-wide screening allowed detection of 12 (7.4%) potentially viable clinically relevant chromosomal abnormalities, which would have remained overlooked if only conventional NIPT had been performed. This resulted in a statistically significant higher sensitivity (100% vs 92.64%, p < 0.001) than did standard screening. This was achieved without sacrificing the specificity of the test, which resulted similar to that obtained with standard cfDNA testing (99.87% vs 99.77%, p = 0.064). CONCLUSION: Genome-wide cfDNA analysis represents an enhanced screening tool for prenatal detection of chromosomal abnormalities, allowing identification of clinically relevant imbalances that are not detectable by conventional cfDNA testing. The results of this study demonstrate the clinical utility of genome-wide cfDNA analysis. This level of screening provides a significant higher sensitivity compared to standard screening while maintaining a high specificity, with the potential to improve overall pregnancy management. © 2017 John Wiley & Sons, Ltd.

The importance of determining the limit of detection of non-invasive prenatal testing methods.

OBJECTIVE: Several non-invasive prenatal testing (NIPT) methods, which analyze circulating fetal cell-free DNA (cfDNA) in maternal plasma, suggest a fetal fraction (FF) ≥ 4% for a reportable result, with the assumption that fetal aneuploidies may not be detectable at lower FF. This study determined the actual limit of detection (LOD) of a massively parallel sequencing-based NIPT method and evaluated its performance in testing samples with low FF. METHOD: An experimental model, involving the creation of artificial plasma mixtures with a final aneuploid FF ranging from 1% to 4%, simulated samples at different proportions of fetal cfDNA. We then analyzed 7103 blood samples, from pregnant women undergoing NIPT, to assess the impact of low FF on the performance of cfDNA testing. RESULTS: Detection of common aneuploidies in samples with an FF as low as 2% is well within the ability of this technology. Of 105 pregnancies confirmed chromosomally abnormal, 25 (23.8%) involving a 2% < FF < 4% were consistently detected. These high-risk pregnancies would have not been identified using the suggested 4% FF cut-off. CONCLUSION: This study underscores the importance of determining the actual LOD for each specific NIPT methodology. It may reduce the incidence of test cancelations and shorten the time required for the diagnosis of aneuploidy. © 2016 John Wiley & Sons, Ltd.

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.

Comparative genomic hybridization selection of blastocysts for repeated implantation failure treatment: a pilot study.

The aim of this study is to determine if the use of preimplantation genetic screening (PGS) by array comparative genomic hybridization (array CGH) and transfer of a single euploid blastocyst in patients with repeated implantation failure (RIF) can improve clinical results. Three patient groups are compared: 43 couples with RIF for whom embryos were selected by array CGH (group RIF-PGS), 33 couples with the same history for whom array CGH was not performed (group RIF NO PGS), and 45 good prognosis infertile couples with array CGH selected embryos (group NO RIF PGS). A single euploid blastocyst was transferred in groups RIF-PGS and NO RIF PGS. Array CGH was not performed in group RIF NO PGS in which 1-2 blastocysts were transferred. One monoembryonic sac with heartbeat was found in 28 patients of group RIF PGS and 31 patients of group NO RIF PGS showing similar clinical pregnancy and implantation rates (68.3% and 70.5%, resp.). In contrast, an embryonic sac with heartbeat was only detected in 7 (21.2%) patients of group RIF NO PGS. In conclusion, PGS by array CGH with single euploid blastocyst transfer appears to be a successful strategy for patients with multiple failed IVF attempts.

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.

Sequential comprehensive chromosome analysis on polar bodies, blastomeres and trophoblast: insights into female meiotic errors and chromosomal segregation in the preimplantation window of embryo development.

STUDY QUESTION: What is the optimal stage from oocyte through preimplantation embryo development for biopsy and preimplantation genetic screening (PGS) to detect abnormal chromosome segregation patterns in eggs or embryos from advanced maternal age (AMA) patients? SUMMARY ANSWER: Testing at the polar body (PB) stage was the least accurate mainly due to the high incidence of post-zygotic events. This suggests that postponing the time of biopsy to the blastocyst stage of preimplantation embryo development may provide the most reliable results for PGS. WHAT IS KNOWN ALREADY: In the PGS field there is an ongoing debate about the optimal biopsy stage for PGS. This is a result of the lack of understanding of how aneuploidy arises in the human embryo. To date, most of the cytogenetic data obtained during PGS investigations have been derived through the analysis of cells at isolated points in the preimplantation window, thus potentially missing critical information on chromosomal segregation. Understanding the chromosome segregation patterns during preimplantation development holds the potential to significantly increase the success rates of IVF. In this study, a sequential comprehensive chromosome analysis of both the PBs and the corresponding embryos at both the cleavage and the blastocyst stages is presented. STUDY DESIGN, SIZE, DURATION: This is a prospective longitudinal cohort study performed between October 2009 and August 2011 involving 9 infertile couples and 21 sets of complete comprehensive chromosomal screening data, including PB1, PB2, corresponding blastomeres and trophectoderm (TE) samples. PARTICIPANTS/MATERIALS, SETTING, METHODS: Infertile couples undergoing IVF cycles with PGS where the female partner was older than 40 years and with a good response to controlled ovarian stimulation (>10 MII oocytes retrieved) were enrolled into the study. The exclusion criteria were (i) patients presenting with abnormal karyotype; (ii) specific ovarian pathologies including polycystic ovary syndrome, endometriosis grade III or higher and premature ovarian failure and (iii) severe male factor infertility (motile sperm count of <500 000/ml after preparation of a fresh ejaculate). The PBs, blastomere and TE samples were sequentially biopsied and analyzed by array comparative genomic hybridization (aCGH). The analysis of chromosome segregation patterns was performed to infer the origin of aneuploidy and to investigate the diagnostic accuracy of both PB and cleavage-stage PGS strategies. MAIN RESULTS AND THE ROLE OF CHANCE: Twenty-one sets of complete data (PB1/PB2/blastomere/TE) including 84 aCGH experiments showed a pattern of multiple meiotic errors typically caused by sister chromatid separation errors and predominantly arising in the second meiotic division. Twenty-two of the 24 (91.7%) errors in the first meiotic division arose as a consequence of premature sister chromatid predivision. In half of these cases, the second meiotic division resulted in a balancing chromosome segregation event producing a normal female complement for that chromosome in the resulting embryo. Overall, only 62 out of 78 (79.5%) of the abnormal meiotic segregations had errors in the either one or both PBs consistent with the aneuploidies observed in their resulting embryos. Ten of the 21 (47.6%) embryos had aneuploidies other than female meiotic-derived ones, most of which detected on Day 3 and confirmed on Day 5 or 6 of embryo development (20/25) with chromosomal loss being three times more frequent than gains. Notably, as high as 20% of female-derived aneuploidies detected on PBs and confirmed on Day 3 were rescued at the blastocyst stage, mainly as a result of diploidization of trisomic chromosomes. On a per chromosome basis, the sensitivity in predicting blastocyst chromosomal complement was significantly lower for PB approach, 61.7%, compared with blastomeres analysis, 86.4% (P < 0.01). LIMITATIONS, REASONS FOR CAUTION: The study was limited to the analysis of oocytes and embryos from AMA patients. Thus, these findings apply only to this patient group. Comparisons with other patient populations including patients with different indications for PGS should be made in future research. In addition, higher resolution and/or more accurate chromosomal screening tests could be used in future studies to corroborate the current findings. WIDER IMPLICATIONS OF THE FINDINGS: These findings provide critical insights into the mechanisms causing errors during female meiosis and the preimplantation embryo development period to improve the design and treatment outcome of PGS.

Chromosomal microarray analysis as a first-line test in pregnancies with a priori low risk for the detection of submicroscopic chromosomal abnormalities.

In this study, we aimed to explore the utility of chromosomal microarray analysis (CMA) in groups of pregnancies with a priori low risk for detection of submicroscopic chromosome abnormalities, usually not considered an indication for testing, in order to assess whether CMA improves the detection rate of prenatal chromosomal aberrations. A total of 3000 prenatal samples were processed in parallel using both whole-genome CMA and conventional karyotyping. The indications for prenatal testing included: advanced maternal age, maternal serum screening test abnormality, abnormal ultrasound findings, known abnormal fetal karyotype, parental anxiety, family history of a genetic condition and cell culture failure. The use of CMA resulted in an increased detection rate regardless of the indication for analysis. This was evident in high risk groups (abnormal ultrasound findings and abnormal fetal karyotype), in which the percentage of detection was 5.8% (7/120), and also in low risk groups, such as advanced maternal age (6/1118, 0.5%), and parental anxiety (11/1674, 0.7%). A total of 24 (0.8%) fetal conditions would have remained undiagnosed if only a standard karyotype had been performed. Importantly, 17 (0.6%) of such findings would have otherwise been overlooked if CMA was offered only to high risk pregnancies.The results of this study suggest that more widespread CMA testing of fetuses would result in a higher detection of clinically relevant chromosome abnormalities, even in low risk pregnancies. Our findings provide substantial evidence for the introduction of CMA as a first-line diagnostic test for all pregnant women undergoing invasive prenatal testing, regardless of risk factors.

Introducing array comparative genomic hybridization into routine prenatal diagnosis practice: a prospective study on over 1000 consecutive clinical cases.

OBJECTIVE: To assess the feasibility of offering array-based comparative genomic hybridization testing for prenatal diagnosis as a first-line test, a prospective study was performed, comparing the results achieved from array comparative genomic hybridization (aCGH) with those obtained from conventional karyotype. METHOD: Women undergoing amniocentesis or chorionic villus sampling were offered aCGH analysis. A total of 1037 prenatal samples were processed in parallel using both aCGH and G-banding for standard karyotyping. Specimen types included amniotic fluid (89.0%), chorionic villus sampling (9.5%) and cultured amniocytes (1.5%). RESULTS: Chromosomal abnormalities were identified in 34 (3.3%) samples; in 9 out of 34 cases (26.5%) aCGH detected pathogenic copy number variations that would not have been found if only a standard karyotype had been performed. aCGH was also able to detect chromosomal mosaicism at as low as a 10% level. There was complete concordance between the conventional karyotyping and aCGH results, except for 2 cases that were only correctly diagnosed by aCGH. CONCLUSIONS: This study demonstrates that aCGH represents an improved diagnostic tool for prenatal detection of chromosomal abnormalities. Although larger studies are needed, our results provide further evidence on the feasibility of introducing aCGH as a first-line diagnostic test in routine prenatal diagnosis practice.

Specific HIV-1 integrase polymorphisms change their prevalence in untreated versus antiretroviral-treated HIV-1-infected patients, all naive to integrase inhibitors.

OBJECTIVES: To define whether the prevalence of mutations associated with integrase inhibitor (INI) resistance is different in untreated versus antiretroviral-treated HIV-1-infected individuals (all INI naive). METHODS: Gene sequences of the integrase (IN) and reverse transcriptase (RT) obtained from plasma samples of a well-defined cohort of 448 HIV-1-infected individuals (134 drug naive and 314 antiretroviral treated) were analysed. Docking simulations, using RT and IN models, were also performed. RESULTS: Primary mutations and the majority of secondary mutations for raltegravir or elvitegravir were completely absent (or rarely found, <1%) in INI-naive patients, either drug naive or antiretroviral treated. Specific IN polymorphisms increased their frequency in antiretroviral-treated patients, and showed positive associations with specific RT resistance mutations. M154I and V165I IN polymorphisms occurred at a frequency of 6% in untreated patients, reaching 21.3% and 13.4%, respectively, in antiretroviral-treated patients. The mutation M154L, absent in drug-naive patients, was prevalent at 5.7% in antiretroviral-treated patients, and was positively associated with RT resistance mutations F227L and T215Y. Similarly, V165I and G163R mutations were associated with the RT resistance mutations F227L and M230L, respectively, and the T206S polymorphism was associated with the RT resistance mutation L210W. Docking simulations showed several favourable contacts between IN and RT residues. CONCLUSIONS: Overall, results confirm that primary and secondary INI-associated mutations are absent or extremely rare in INI-naive patients. Conversely, a few specific IN polymorphisms found in INI-naive patients increased their frequency in antiretroviral-failing patients and/or are associated with RT resistance mutations. The potential contribution of such polymorphisms to the evolution of resistance under the pressure of INIs needs further investigation.