Genome stability of bovine in vivo-conceived cleavage-stage embryos is higher compared to in vitro-produced embryos.

STUDY QUESTION Is the rate and nature of chromosome instability (CIN) similar between bovine in vivo-derived and in vitro-cultured cleavage-stage embryos? SUMMARY ANSWER There is a major difference regarding chromosome stability of in vivo-derived and in vitro-cultured embryos, as CIN is significantly lower in in vivo-derived cleavage-stage embryos compared to in vitro-cultured embryos. WHAT IS KNOWN ALREADY CIN is common during in vitro embryogenesis and is associated with early embryonic loss in humans, but the stability of in vivo-conceived cleavage-stage embryos remains largely unknown. STUDY DESIGN, SIZE, DURATION Because human in vivo preimplantation embryos are not accessible, bovine (Bos taurus) embryos were used to study CIN in vivo. Five young, healthy, cycling Holstein Friesian heifers were used to analyze single blastomeres of in vivo embryos, in vitro embryos produced by ovum pick up with ovarian stimulation (OPU-IVF), and in vitro embryos produced from in vitro matured oocytes retrieved without ovarian stimulation (IVM-IVF). PARTICIPANTS/MATERIALS, SETTING, METHODS Single blastomeres were isolated from embryos, whole-genome amplified and hybridized on Illumina BovineHD BeadChip arrays together with the bulk DNA from the donor cows (mothers) and the bull (father). DNA was also obtained from the parents of the bull and from the parents of the cows (paternal and maternal grandparents, respectively). Subsequently, genome-wide haplotyping and copy-number profiling was applied to investigate the genomic architecture of 171 single bovine blastomeres of 16 in vivo, 13 OPU-IVF and 13 IVM-IVF embryos. MAIN RESULTS AND THE ROLE OF CHANCE The genomic stability of single blastomeres in both of the in vitro-cultured embryo cohorts was severely compromised (P < 0.0001), and the frequency of whole chromosome or segmental aberrations was higher in embryos produced in vitro than in embryos derived in vivo. Only 18.8% of in vivo-derived embryos contained at least one blastomere with chromosomal anomalies, compared to 69.2% of OPU-IVF embryos (P < 0.01) and 84.6% of IVM-IVF embryos (P < 0.001). LARGE SCALE DATA Genotyping data obtained in this study has been submitted to NCBI Gene Expression Omnibus (GEO; accession number GSE95358) LIMITATIONS REASONS FOR CAUTION There were two main limitations of the study. First, animal models may not always reflect the nature of human embryogenesis, although the use of an animal model to investigate CIN was unavoidable in our study. Second, a limited number of embryos were obtained, therefore more studies are warranted to corroborate the findings. WIDER IMPLICATIONS OF THE FINDINGS Although CIN is also present in in vivo-developed embryos, in vitro procedures exacerbate chromosomal abnormalities during early embryo development. Hence, the present study highlights that IVF treatment compromises embryo viability and should be applied with care. Additionally, our results encourage to refine and improve in vitro culture conditions and assisted reproduction technologies. STUDY FUNDING/COMPETING INTEREST(S) The study was funded by the Agency for Innovation by Science and Technology (IWT) (TBM-090878 to J.R.V. and T.V.), the Research Foundation Flanders (FWO; G.A093.11 N to T.V. and J.R.V. and G.0392.14 N to A.V.S. and J.R.V.), the European Union's FP7 Marie Curie Industry-Academia Partnerships and Pathways (IAPP, SARM, EU324509 to J.R.V., T.V., O.T, A.D., A.S. and A.K.) and Horizon 2020 innovation programme (WIDENLIFE, 692065 to J.R.V., O.T., T.V., A.K. and A.S.). M.Z.E., J.R.V. and T.V. are co-inventors on a patent application ZL913096-PCT/EP2014/068315-WO/2015/028576 (‘Haplotyping and copy-number typing using polymorphic variant allelic frequencies’), licensed to Cartagenia (Agilent Technologies

Genome-wide haplotyping embryos developing from 0PN and 1PN zygotes increases transferrable embryos in PGT-M.

STUDY QUESTION Can genome-wide haplotyping increase success following preimplantation genetic testing for a monogenic disorder (PGT-M) by including zygotes with absence of pronuclei (0PN) or the presence of only one pronucleus (1PN)? SUMMARY ANSWER Genome-wide haplotyping 0PNs and 1PNs increases the number of PGT-M cycles reaching embryo transfer (ET) by 81% and the live-birth rate by 75%. WHAT IS KNOWN ALREADY Although a significant subset of 0PN and 1PN zygotes can develop into balanced, diploid and developmentally competent embryos, they are usually discarded because parental diploidy detection is not part of the routine work-up of PGT-M. STUDY DESIGN, SIZE, DURATION This prospective cohort study evaluated the pronuclear number in 2229 zygotes from 2337 injected metaphase II (MII) oocytes in 268 cycles. PGT-M for 0PN and 1PN embryos developing into Day 5/6 blastocysts with adequate quality for vitrification was performed in 42 of the 268 cycles (15.7%). In these 42 cycles, we genome-wide haplotyped 216 good quality embryos corresponding to 49 0PNs, 15 1PNs and 152 2PNs. The reported outcomes include parental contribution to embryonic ploidy, embryonic aneuploidy, genetic diagnosis for the monogenic disorder, cycles reaching ETs, pregnancy and live birth rates (LBR) for unaffected offspring. PARTICIPANTS/MATERIALS, SETTING, METHODS Blastomere DNA was whole-genome amplified and hybridized on the Illumina Human CytoSNP12V2.1.1 BeadChip arrays. Subsequently, genome-wide haplotyping and copy-number profiling was applied to investigate the embryonic genome architecture. Bi-parental, unaffected embryos were transferred regardless of their initial zygotic PN score. MAIN RESULTS AND THE ROLE OF CHANCE A staggering 75.51% of 0PN and 42.86% of 1PN blastocysts are diploid bi-parental allowing accurate genetic diagnosis for the monogenic disorder. In total, 31% (13/42) of the PGT-M cycles reached ET or could repeat ET with an unaffected 0PN or 1PN embryo. The LBR per initiated cycle increased from 9.52 to 16.67%. LIMITATIONS, REASONS FOR CAUTION The clinical efficacy of the routine inclusion of 0PN and 1PN zygotes in PGT-M cycles should be confirmed in larger cohorts from multicenter studies. WIDER IMPLICATIONS OF THE FINDINGS Genome-wide haplotyping allows the inclusion of 0PN and 1PN embryos and subsequently increases the cycles reaching ET following PGT-M and potentially PGT for aneuploidy (PGT-A) and chromosomal structural rearrangements (PGT-SR). Establishing measures of clinical efficacy could lead to an update of the ESHRE guidelines which advise against the use of these zygotes. STUDY FUNDING/COMPETING INTEREST(S) SymBioSys (PFV/10/016 and C1/018 to J.R.V. and T.V.), the Horizon 2020 WIDENLIFE: 692065 to J.R.V., T.V., E.D., A.D. and M.Z.E. M.Z.E., T.V. and J.R.V. co-invented haplarithmisis (‘Haplotyping and copy-number typing using polymorphic variant allelic frequencies’), which has been licensed to Agilent Technologies. H.M. is fully supported by the (FWO) (ZKD1543-ASP/16). The authors have no competing interests to declare

Preimplantation genetic testing for Neurofibromatosis type 1:more than 20 years of clinical experience

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder that affects the skin and the nervous system. The condition is completely penetrant with extreme clinical variability, resulting in unpredictable manifestations in affected offspring, complicating reproductive decision-making. One of the reproductive options to prevent the birth of affected offspring is preimplantation genetic testing (PGT). We performed a retrospective review of the medical files of all couples (n = 140) referred to the Dutch PGT expert center with the indication NF1 between January 1997 and January 2020. Of the couples considering PGT, 43 opted out and 15 were not eligible because of failure to identify the underlying genetic defect or unmet criteria for in vitro fertilization (IVF) treatment. The remaining 82 couples proceeded with PGT. Fertility assessment prior to IVF treatment showed a higher percentage of male infertility in males affected with NF1 compared to the partners of affected females. Cardiac evaluations in women with NF1 showed no contraindications for IVF treatment or pregnancy. For 67 couples, 143 PGT cycles were performed. Complications of IVF treatment were not more prevalent in affected females compared to partners of affected males. The transfer of 174 (out of 295) unaffected embryos led to 42 ongoing pregnancies with a pregnancy rate of 24.1% per embryo transfer. There are no documented cases of misdiagnosis following PGT in this cohort. With these results, we aim to provide an overview of PGT for NF1 with regard to success rate and safety, to optimize reproductive counseling and PGT treatment for NF1 patients.</p

Single-cell paired-end genome sequencing reveals structural variation per cell cycle

The nature and pace of genome mutation is largely unknown. Because standard methods sequence DNA from populations of cells, the genetic composition of individual cells is lost, de novo mutations in cells are concealed within the bulk signal and per cell cycle mutation rates and mechanisms remain elusive. Although single-cell genome analyses could resolve these problems, such analyses are error-prone because of whole-genome amplification (WGA) artefacts and are limited in the types of DNA mutation that can be discerned. We developed methods for paired-end sequence analysis of single-cell WGA products that enable (i) detecting multiple classes of DNA mutation, (ii) distinguishing DNA copy number changes from allelic WGA-amplification artefacts by the discovery of matching aberrantly mapping read pairs among the surfeit of paired-end WGA and mapping artefacts and (iii) delineating the break points and architecture of structural variants. By applying the methods, we capture DNA copy number changes acquired over one cell cycle in breast cancer cells and in blastomeres derived from a human zygote after in vitro fertilization. Furthermore, we were able to discover and fine-map a heritable inter-chromosomal rearrangement t(1;16)(p36;p12) by sequencing a single blastomere. The methods will expedite applications in basic genome research and provide a stepping stone to novel approaches for clinical genetic diagnosis

In vitro fertilization does not increase the incidence of de novo copy number alterations in fetal and placental lineages

Although chromosomal instability (CIN) is a common phenomenon in cleavage-stage embryogenesis following in vitro fertilization (IVF)1,2,3, its rate in naturally conceived human embryos is unknown. CIN leads to mosaic embryos that contain a combination of genetically normal and abnormal cells, and is significantly higher in in vitro-produced preimplantation embryos as compared to in vivo-conceived preimplantation embryos4. Even though embryos with CIN-derived complex aneuploidies may arrest between the cleavage and blastocyst stages of embryogenesis5,6, a high number of embryos containing abnormal cells can pass this strong selection barrier7,8. However, neither the prevalence nor extent of CIN during prenatal development and at birth, following IVF treatment, is well understood. Here we profiled the genomic landscape of fetal and placental tissues postpartum from both IVF and naturally conceived children, to investigate the prevalence and persistence of large genetic aberrations that probably arose from IVF-related CIN. We demonstrate that CIN is not preserved at later stages of prenatal development, and that de novo numerical aberrations or large structural DNA imbalances occur at similar rates in IVF and naturally conceived live-born neonates. Our findings affirm that human IVF treatment has no detrimental effect on the chromosomal constitution of fetal and placental lineages

Noninvasive Prenatal Test Results Indicative of Maternal Malignancies:A Nationwide Genetic and Clinical Follow-Up Study

PURPOSE: Noninvasive prenatal testing (NIPT) for fetal aneuploidy screening using cell-free DNA derived from maternal plasma can incidentally raise suspicion for cancer. Diagnostic routing after malignancy suspicious-NIPT faces many challenges. Here, we detail malignancy suspicious-NIPT cases, and describe the clinical characteristics, chromosomal aberrations, and diagnostic routing of the patients with a confirmed malignancy. Clinical lessons can be learned from our experience. METHODS: Patients with NIPT results indicative of a malignancy referred for tumor screening between April 2017 and April 2020 were retrospectively included from a Dutch nationwide NIPT implementation study, TRIDENT-2. NIPT profiles from patients with confirmed malignancies were reviewed, and the pattern of chromosomal aberrations related to tumor type was analyzed. We evaluated the diagnostic contribution of clinical and genetic examinations. RESULTS: Malignancy suspicious-NIPT results were reported in 0.03% after genome-wide NIPT, and malignancies confirmed in 16 patients (16/48, 33.3%). Multiple chromosomal aberrations were seen in 23 of 48 patients with genome-wide NIPT, and a malignancy was confirmed in 16 patients (16/23, 69.6%). After targeted NIPT, 0.005% malignancy suspicious-NIPT results were reported, in 2/3 patients a malignancy was confirmed. Different tumor types and stages were diagnosed, predominantly hematologic malignancies (12/18). NIPT data showed recurrent gains and losses in primary mediastinal B-cell lymphomas and classic Hodgkin lymphomas. Magnetic resonance imaging and computed tomography were most informative in diagnosing the malignancy. CONCLUSION: In 231,896 pregnant women, a low percentage (0.02%) of NIPT results were assessed as indicative of a maternal malignancy. However, when multiple chromosomal aberrations were found, the risk of a confirmed malignancy was considerably high. Referral for extensive oncologic examination is recommended, and may be guided by tumor-specific hallmarks in the NIPT profile

Haplarithmisis to study haplotypes genome-wide to single-cell resolution, enabling a generic method for preimplantation genectic diagnosis in the clinic and novel fundamental genome research.

Prior to the inception of my PhD research, there was a need for a new generation of genome analysis methods having the capacity of reconstructing haplotypes and determining the copy number state of those haplotypes in a DNA sample of a single cell or multiple cells. Such a method was needed to enable a generic mode of preimplantation genetic diagnosis of human embryos following in vitro fertilization in the clinic and to develop novel understanding of genome instability and plasticity in different developmental stages of life. To develop such a method for genome-wide haplotyping and copy number profiling of single cells that overcomes the impediments associated with genotype errors due to whole genome amplification (WGA) artifacts as well as non-diploid copy number states of SNPs, we devised a novel concept, termed haplarithmisis (Greek for haplotype numbering), which deciphers single-cell SNP B-allele frequencies on the basis of phased parental genotypes, to determine haplotypes, copy number information about the haplotypes as well as the parental and segregational origin of putative haplotype anomalies in the cell. We demonstrated that haplarithmisis leverages conventional methods for genome-wide haplotyping as well as DNA copy number profiling. We embedded haplarithmisis in a broad computational algorithmic suite, called siCHILD, which is an abbreviation of ‘single-cell haplotyping and imputation of linked disease variants’, and which consists of five innovative modules, including (1) quality control of single-cell SNP data, (2) haplarithmisis using single-cell SNP B-allele frequencies, (3) haplotyping using discrete single-cell SNP-calls (AA, AB, BB), (4) copy number analysis of the haplotypes in the cell, and (5) the visualization of the data. We benchmarked this methodology with SNP-array data of human single lymphoblastoid cells containing known haplotype architectures from bulk DNA analyses. Subsequently, we applied the methodology to single blastomeres of human embryos to infer the inheritance of Mendelian disease-causing genetic variants transmitted from the parent(s) to the embryo using linkage to neighboring SNPs in a haplotype, as well as to further characterize the nature of chromosome instability in human embryos. These results were independently confirmed by locus-specific FISH-, PCR- or sequencing-based genetic diagnoses on separate cells of the same embryos in the clinic as well as single-cell genome sequencing, delivering a comprehensive clinical validation of the method for diagnosing the transmission of genetic variants causing autosomal dominant and recessive disorders and X-linked disorders, as well as the inheritance of derivative chromosomes of parental reciprocal translocations. By applying haplarithmisis, we were able to observe genomic constitutions to which current state-of-the-art single-cell haplotyping and DNA-copy number profiling methods are blind. For instance, a single human blastomere with a tetraploid haplotype constitution and 6 trisomic chromosomes could be distinguished from a diploid cell with monosomies for the same chromosomes. In addition, we could discern meiotic trisomies from mitotic ones. Furthermore, we also applied haplarithmsis on bovine cleavage stage embryos and observed a frequency of chromosome instability that is similar to human cleavage stage embryos following in vitro fertilization. However, using haplarithmisis we discovered an extraordinary amount of mixoploid and/or chimeric cell lineages co-existing in the same bovine embryo and discuss their mechanistic origin. In addition, we proved that haplarithmisis has a broad applicative value not only by enabling concurrent haplotyping and copy number typing of single cells but also by unraveling the mosaic and chimeric allelic architectures of multi-cell DNA samples. This may have a huge impact on deciphering the etiology of genomic disorders and developing understanding of the mechanistic origin of the genetic anomalies. In conclusion, we developed, applied and translated an ensemble of innovative genome analysis approaches that uncovers the haplotype architecture of entire genomes in DNA derived from many cells down to a single cell, demonstrating novel applications in the clinic and fundamental genome research.nrpages: 285status: publishe

Haplotyping and copy-number profiling of single cells by massive parallel sequencing

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Non-allelic homologous recombination between retrotransposable elements is a driver of de novo unbalanced translocations

Large scale analysis of balanced chromosomal translocation breakpoints has shown nonhomologous end joining and microhomology-mediated repair to be the main drivers of interchromosomal structural aberrations. Breakpoint sequences of de novo unbalanced translocations have not yet been investigated systematically. We analyzed 12 de novo unbalanced translocations and mapped the breakpoints in 9. Surprisingly, in contrast to balanced translocations, we identify non-allelic homologous recombination (NAHR) between (retro)transposable elements and especially long interspersed elements (LINEs) as the main mutational mechanism. This finding shows yet another involvement of (retro)transposons in genomic rearrangements and exposes a profoundly different mutational mechanism compared to balanced chromosomal translocations. Furthermore, we show the existence of compound maternal/paternal derivative chromosomes, reinforcing the hypothesis that human cleavage stage embryogenesis is a cradle of chromosomal rearrangements.status: publishe

Zygotes segregate entire parental genomes in distinct blastomere lineages causing cleavage-stage chimerism and mixoploidy

Dramatic genome dynamics, such as chromosome instability, contribute to the remarkable genomic heterogeneity among the blastomeres comprising a single embryo during human preimplantation development. This heterogeneity, when compatible with life, manifests as constitutional mosaicism, chimerism, and mixoploidy in live-born individuals. Chimerism and mixoploidy are defined by the presence of cell lineages with different parental genomes or different ploidy states in a single individual, respectively. Our knowledge of their mechanistic origin results from indirect observations, often when the cell lineages have been subject to rigorous selective pressure during development. Here, we applied haplarithmisis to infer the haplotypes and the copy number of parental genomes in 116 single blastomeres comprising entire preimplantation bovine embryos (n = 23) following in vitro fertilization. We not only demonstrate that chromosome instability is conserved between bovine and human cleavage embryos, but we also discovered that zygotes can spontaneously segregate entire parental genomes into different cell lineages during the first post-zygotic cleavage division. Parental genome segregation was not exclusively triggered by abnormal fertilizations leading to triploid zygotes, but also normally fertilized zygotes can spontaneously segregate entire parental genomes into different cell lineages during cleavage of the zygote. We coin the term "heterogoneic division" to indicate the events leading to noncanonical zygotic cytokinesis, segregating the parental genomes into distinct cell lineages. Persistence of those cell lines during development is a likely cause of chimerism and mixoploidy in mammals