Diego Marin

Preclinical validation of a targeted next generation sequencing-based comprehensive chromosome screening methodology in human blastocysts

STUDY QUESTION Can a novel targeted next generation sequencing (tNGS) platform accurately detect whole chromosome aneuploidy in a trophectoderm biopsy and provide additional information to improve testing? SUMMARY ANSWER Karyotypes obtained by tNGS were concordant with other validated platforms and single nucleotide polymorphism genotyping information obtained can be used for improved detection and quality control. WHAT IS KNOWN ALREADY qPCR-based whole chromosome aneuploidy screening is highly accurate in comparison to other common methods and has been shown to improve IVF success in two randomized clinical trials. With aneuploidy screening becoming standard of care in many IVF centres, there is a need to develop platforms with high throughput, low cost capabilities. STUDY DESIGN SIZE, DURATION Twelve well-characterized cell lines were obtained from a commercial cell line repository and 31 discarded human blastocysts were obtained from 17 IVF patients who underwent comprehensive chromosome screening (CCS). PARTICIPANTS/MATERIAL, SETTING, METHODS All samples were processed using a unique amplification strategy which directly incorporated sequencing library adapters and barcodes. Sequencing was performed on an Ion Torrent Proton. A custom bioinformatics pipeline was used to determine the karyotype for each sample. The consistency of tNGS diagnoses with either conventional karyotyping of cell lines or quantitative real-time PCR based CCS of blastocyst biopsies was evaluated. MAIN RESULTS AND THE ROLE OF CHANCE Overall consistency per sample of tNGS based CCS in 5-cell samples from a variety of cell lines was 99.2%. In the blinded analysis of rebiopsies of aneuploid blastocysts, an overall targeted tNGS CCS consistency of 98.7% was observed per sample. These data demonstrate the ability of tNGS based CCS to provide an accurate and high throughput evaluation of aneuploidy in the human blastocyst. LARGE SCALE DATA Not applicable. LIMITATIONS REASONS FOR CAUTION This study is limited to whole chromosome aneuploidy, as mosaicism and segmental aneuploidy have not been investigated. WIDER IMPLICATIONS OF THE FINDINGS These data show an accurate, high throughput method, and with the greater depth of each amplicon sequenced in comparison to commercial kits, there is greater application available for single nucleotide polymorphism based analysis for quality control. STUDY FUNDING/COMPETING INTERESTS This study was funded through intramural research funds provided by the Foundation for Embryonic Competence. There are no competing interests.

Identification and characterization of Aurora kinase B and C variants associated with maternal aneuploidy

STUDY QUESTION Are single nucleotide variants (SNVs) in Aurora kinases B and C (AURKB, AURKC) associated with risk of aneuploid conception? SUMMARY ANSWER Two SNVs were found in patients with extreme aneuploid concepti rates with respect to their age; one variant, AURKC p.I79V, is benign, while another, AURKB p.L39P, is a potential gain-of-function mutant with increased efficiency in promoting chromosome alignment. WHAT IS KNOWN ALREADY Maternal age does not always predict aneuploidy risk, and rare gene variants can be drivers of disease. The AURKB and AURKC regulate chromosome segregation, and are associated with reproductive impairments in mouse and human. STUDY DESIGN, SIZE, DURATION An extreme phenotype sample selection scheme was performed for variant discovery. Ninety-six DNA samples were from young patients with higher than average embryonic aneuploidy rates and an additional 96 DNA samples were from older patients with lower than average aneuploidy rates. PARTICIPANTS/MATERIALS, SETTING, METHODS Using the192 DNA samples, the coding regions of AURKB and AURKC were sequenced using next generation sequencing. To assess biological significance, we expressed complementary RNA encoding the human variants in mouse oocytes. Assays such as determining subcellular localization and assessing catalytic activity were performed to determine alterations in protein function during meiosis. MAIN RESULTS AND THE ROLE OF CHANCE Ten SNVs were identified using three independent variant-calling methods. Two of the SNVs (AURKB p.L39P and AURKC p.I79V) were non-synonymous and identified by at least two variant-identification methods. The variant encoding AURKC p.I79V, identified in a young woman with a higher than average rate of aneuploid embryos, showed wild-type localization pattern and catalytic activity. On the other hand, the variant encoding AURKB p.L39P, identified in an older woman with lower than average rates of aneuploid embryos, increased the proteins ability to regulate alignment of chromosomes at the metaphase plate. These experiments were repeated three independent times using 2-3 mice for each trial. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Biological significance of the human variants was assessed in an in vitro mouse oocyte model where the variants are over-expressed. Therefore, the human protein may not function identically to the mouse homolog, or the same in mouse oocytes as in human oocytes. Furthermore, supraphysiological expression levels may not accurately reflect endogenous activity. Moreover, the evaluated variants were identified in one patient each, and no trial linking the SNV to pregnancy outcomes was conducted. Finally, the patient aneuploidy rates were established by performing comprehensive chromosome screening in blastocysts, and because of the link between female gamete aneuploidy giving rise to aneuploid embryos, we evaluate the role of the variants in Meiosis I. However, it is possible that the chromosome segregation mistake arose during Meiosis II or in mitosis in the preimplantation embryo. Their implications in human female meiosis and aneuploidy risk remain to be determined. WIDER IMPLICATIONS OF THE FINDINGS The data provide evidence that gene variants exist in reproductively younger or advanced aged women that are predictive of the risk of producing aneuploid concepti in humans. Furthermore, a single amino acid in the N-terminus of AURKB is a gain-of-function mutant that could be protective of euploidy. STUDY FUNDING/COMPETING INTERESTS This work was supported by a Research Grant from the American Society of Reproductive Medicine and support from the Charles and Johanna Busch Memorial Fund at Rutgers, the State University of NJ to K.S. and the Foundation for Embryonic Competence, Inc to N.T. The authors declare no conflicts of interest.

Preimplantation embryonic mosaicism: origin, consequences and the reliability of comprehensive chromosome screening.

Purpose of review Embryonic mosaicism represents an ongoing challenge for contemporary comprehensive chromosome screening platforms due to the unknown reproductive potential of mosaic embryos and technical difficulties of its detection from a single embryo biopsy. Recent findings Mosaicism in preimplantation embryos is a product of mitotic errors arising primarily from anaphase lag and chromosome nondisjunction. To date, there is high variability among estimations of prevalence of mosaicism in blastocysts, the most recent ranging from 3.3 to 83%. It has been reported that alleged mosaic embryos can develop into healthy babies, although the proper study evaluating this question remains to be completed. Technical artefacts from comprehensive chromosome screening platforms may also hinder correct classification of embryos as genuine mosaics. Summary Although complex, embryonic mosaicism is a phenomenon that deserves further investigation. Many embryos classified as mosaic may have actual reproductive potential. The predictive value of intermediate chromosome copy number assignments for the remaining embryo and for ongoing reproductive potential needs more careful consideration. In addition, recent advancements in extended embryo culture raise the possibility of investigating whether preferential segregation, selective advantage of normal cells or surveillance of abnormal chromosome numbers occur at postimplantation stages.

Validation of a targeted next generation sequencing-based comprehensive chromosome screening platform for detection of triploidy in human blastocysts

Triploidy accounts for ~2% of natural pregnancies and 15% of cytogenetically abnormal miscarriages. This study aimed to validate triploidy detection in human blastocysts, its frequency and parental origin using genotyping data generated in parallel with chromosome copy number analysis by a targeted next generation sequencing (tNGS)-based comprehensive chromosome screening platform. Phase 1: diploid and triploid control samples were blinded, sequenced by tNGS and karyotype predictions compared for accuracy. Phase 2: tNGS was used to calculate the frequency of triploidy in 18,791 human blastocysts from trophectoderm (TE) biopsies. Phase 3: parental origin of the inherited extra alleles was evaluated by sequencing parental gDNA to validate triploidy predictions from Phase 2. All karyotypes and ploidy in controls from Phase 1 were correctly predicted by two independent methods. A blastocyst triploidy frequency of 0.474% (89/18,791) was observed in Phase 2 of the study. Finally, five suspected triploid blastocysts with parental DNA available were confirmed to be triploid and of maternal origin. tNGS provides higher sequencing depth in contrast to other contemporary NGS platforms, allowing for accurate single nucleotide polymorphism calling and accurate detection of triploidy in TE biopsies. Triploidy in intracytoplasmic sperm injection-derived blastocysts is rare and mostly of maternal origin.

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

Comprehensive chromosome screening and gene expression analysis from the same biopsy in human preimplantation embryos

Abstract STUDY QUESTION Can simultaneous comprehensive chromosome screening (CCS) and gene expression analysis be performed on the same biopsy of preimplantation human embryos? SUMMARY ANSWER For the first time, CCS and reliable gene expression analysis have been performed on the same human preimplantation embryo biopsy. WHAT IS KNOWN ALREADY A single trophectoderm (TE) biopsy is routinely used for many IVF programs offering CCS for selection of only chromosomally normal embryos for transfer. Although the gene expression profiling of human preimplantation embryos has been described, to date no protocol allows for simultaneous CCS and gene expression profiling from a single TE biopsy. STUDY DESIGN, SIZE AND DURATION This is a proof of concept and validation study structured in two phases. In Phase 1, cell lines were subjected to a novel protocol for combined CCS and gene expression analysis so as to validate the accuracy and reliability of the proposed protocol. In Phase 2, 20 donated human blastocysts were biopsied and processed with the proposed protocol in order to obtain an accurate CCS result and characterize their gene expression profiles using the same starting material. PARTICIPANTS/MATERIALS, SETTING AND METHOD A novel protocol coupling quantitative real-time PCR-based CCS and gene expression analysis using RT-PCR was designed for this study. Phase 1: six-cell aliquots of well-characterized fibroblast cell lines (GM00323, 46,XY and GM04435, 48,XY,+16,+21) were subjected to the proposed protocol. CCS results were compared with the known karyotypes for consistency, and gene expression levels were compared with levels of purified RNA from same cell lines for validation of reliable gene expression profiling. Phase 2: four biopsies were performed on 20 frozen human blastocysts previously diagnosed as trisomy 21 (10 embryos) and monosomy 21 (10 embryos) by CCS. All samples were processed with the proposed protocol and re-evaluated for concordance with the original CCS result. Their gene expression profiles were characterized and differential gene expression among embryos and early embryonic cell lineages was also evaluated. MAIN RESULTS AND THE ROLE OF CHANCE CCS results from cell lines showed 100% consistency with their known karyotypes. ΔΔCt values of differential gene expression of four selected target genes from the cell lines GM4435 and GM0323 were comparable between six-cell aliquots and purified RNA (Collagen type I alpha-1 (COL1A1), P = 0.54; Fibroblast growth factor-5 (FGF5), P = 0.11; Laminin subunit beta-1 (LAMB1), P = 1.00 and Atlastin-1 (ATL1), P = 0.23). With respect to human blastocysts, 92% consistency was reported after comparing embryonic CCS results with previous diagnosis. A total of 30 genes from a human stem cell pluripotency panel were selected to evaluate gene expression in human embryos. Correlation coefficients of expression profiles from biopsies of the same embryo (r = 0.96 ± 0.03 (standard deviation), n = 45) were significantly higher than when biopsies from unrelated embryos were evaluated (r = 0.93 ± 0.03, n = 945) (P < 0.0001). Growth differentiation factor 3 (GDF3) was found to be significantly up-regulated in the inner cell mass (ICM), whereas Caudal type homebox protein-2 (CDX2), Laminin subunit alpha-1 (LAMA1) and DNA methyltransferase 3-beta (DNMT3B) showed down-regulation in ICM compared with TE. Trisomy 21 embryos showed significant up-regulation of markers of cell differentiation (Cadherin-5 (CDH5) and Laminin subunit gamma-1 (LAMC1)), whereas monosomy 21 blastocysts showed higher expression of genes reported to be expressed in undifferentiated cells (Gamma-Aminobutyric Acid Type-A Receptor Beta3 Subunit (GABRB3) and GDF3). LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION Gene expression profiles of chromosomally normal embryos were not assessed due to restrictive access to euploid embryos for research. Nonetheless, the profile of blastocysts with single aneuploidies was characterized and compared. Only 30 target genes were analyzed for gene expression in this study. Increasing the number of target genes will provide a more comprehensive transcriptomic signature and reveal potential pathways paramount for embryonic competence and correct development. WIDER IMPLICATIONS OF THE FINDINGS This is the first time that CCS and gene expression analysis have been performed on the same human preimplantation embryo biopsy. Further optimization of this protocol with other CCS platforms and inclusion of more target genes will provide innumerable research and clinical applications, such as discovery of biomarkers for embryonic reproductive potential and characterization of the transcriptomic signatures of embryos, potentially allowing for further embryo selection prior to embryo transfer and therefore improving outcomes. STUDY FUNDING AND COMPETING INTERESTS This study was funded by the Foundation for Embryonic Competence, Basking Ridge, NJ, USA. No conflicts of interests declared.