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

Parental genomes segregate into distinct blastomeres during multipolar zygotic divisions leading to mixoploid and chimeric blastocysts

BACKGROUND: During normal zygotic division, two haploid parental genomes replicate, unite and segregate into two biparental diploid blastomeres. RESULTS: Contrary to this fundamental biological tenet, we demonstrate here that parental genomes can segregate to distinct blastomeres during the zygotic division resulting in haploid or uniparental diploid and polyploid cells, a phenomenon coined heterogoneic division. By mapping the genomic landscape of 82 blastomeres from 25 bovine zygotes, we show that multipolar zygotic division is a tell-tale of whole-genome segregation errors. Based on the haplotypes and live-imaging of zygotic divisions, we demonstrate that various combinations of androgenetic, gynogenetic, diploid, and polyploid blastomeres arise via distinct parental genome segregation errors including the formation of additional paternal, private parental, or tripolar spindles, or by extrusion of paternal genomes. Hence, we provide evidence that private parental spindles, if failing to congress before anaphase, can lead to whole-genome segregation errors. In addition, anuclear blastomeres are common, indicating that cytokinesis can be uncoupled from karyokinesis. Dissociation of blastocyst-stage embryos further demonstrates that whole-genome segregation errors might lead to mixoploid or chimeric development in both human and cow. Yet, following multipolar zygotic division, fewer embryos reach the blastocyst stage and diploidization occurs frequently indicating that alternatively, blastomeres with genome-wide errors resulting from whole-genome segregation errors can be selected against or contribute to embryonic arrest. CONCLUSIONS: Heterogoneic zygotic division provides an overarching paradigm for the development of mixoploid and chimeric individuals and moles and can be an important cause of embryonic and fetal arrest following natural conception or IVF

Chromosomal aberrations, epigentic factors and retrotransposition mechanisms

We have studied the RNA expression levels and retrotrasposition potential of HERV-K10 in cancer cell lines (HeLa, A549 and H1299) and immature human oocytes. We have detected high levels of expressed HERV-K10 RNA and constructed truncated recombinant retroviruses tagged with an EGFP gene-based retrotransposition cassette for the detection of retrotransposition events. Cancer cell lines were transfected and immature human oocytes were microinjected with the recombinant construct. We detected retrotransposition events through EGFP-positive cells, further confirmed in the genome by PCR analysis. Using FACS, measured retrotransposition frequency was found at high levels up to 38.3%. High retrotransposition levels are related to phenotypic changes of the cells, cell cycle disturbance with cease of cells in G2/M phase and multiple chromosomal breaks. We have also shown that retrotransposition events take place due to the expression of a HERV-K10-specific RT encoded by endogenous elements. Our findings demonstrate for the first time that endogenous retrovirus HERV-K10 is retrotransposition-competent, even if truncated/mutated, and that cancer and developing germ cells provide a cellular environment favorable for generation of retrotransposition events. Next, we have studied DNA methylation patterns of HERV-K10 retrotransposon and DLK1/MEG3, CDKN1C (p57KIP2) and IGF2/H19 imprinted genes using MS-PCR in CVs from children conceived after IVF-PGD and testis biopsies and sperm samples from infertile men. In the PGD group, MS-PCR analysis revealed a statistically significant alteration of HERV-K10 and DLK1/MEG3 methylation pattern compared to controls. Aberrant methylation patterns were found in the paternally imprinted genes tested in biopsy samples. In sperm samples, there has been observed a mild aberration of HERV-K10, while the severity of the aberration of the imprinted genes tested was as follows: DLK1/MEG3>IGF2/H19>CDKN1C (p57KIP2). Sequence analysis of the imprinted genes studied using bioinformatics uncovered high content in transposable element derived sequences was high in the case of DLK1/MEG3, quite low for IGF2/H19 and even lower for CDKN1C (p57KIP2). Consequently, profound aberration of the methylation patterns of paternally imprinted genes are possibly associated with severe infertility and simultaneous aberration of DLK1/MEG3 and IGF2/H19 could be used as sperm quality marker. Our results suggest that altered HERV-K10 methylation is correlated to imprinting alterations in a retrotransposon-rich genomic region, and that retrotransposons may play a role in the regulation of imprinting.Μελετήσαμε την ικανότητα έκφρασης RNA και ρετρομετάθεσης του ρετροϊού HERV-K10 σε κύτταρα HeLa, A549 και H1299 και σε ανθρώπινα ωοκύτταρα. Ανιχνεύσαμε έκφραση RNA του σε υψηλά επίπεδα και κατασκευάσαμε ανασυνδυασμένους ελλειμματικούς ρετροϊούς σημασμένους με ειδική κασέτα ανίχνευσης της ρετρομετάθεσης που βασίζεται στην έκφραση της φθορισμογόνου πρωτεΐνης EGFP. Μετά από διαμόλυνση καρκινικών κυτταρικών σειρών με αυτούς και μικροένεσή τους σε άωρα ανθρώπινα ωοκύτταρα, μέσω μικροσκοπίας UV ανιχνεύσαμε γεγονότα ρετρομετάθεσης, τα οποία πιστοποιήθηκαν σε επίπεδο γονιδιώματος με PCR, ενώ επιπλέον με FACS μετρήθηκε η συχνότητα ρετρομεταθέσεων σε υψηλά επίπεδα ως και 38.3%. Υψηλή συχνότητα ρετρομετάθεσης συσχετίζεται με έντονες φαινοτυπικές αλλαγές, διαταραχές του κυτταρικού κύκλου με συσσώρευση κυττάρων στη G2/M και πολλαπλές θραύσεις στα χρωμοσώματα. Αποδείξαμε ότι τα γεγονότα ρετρομετάθεσης συμβαίνουν χάρη στην εξειδικευμένη δράση αντίστροφης μεταγραφάσης ειδικής για τα HERV-K10 που κωδικοποιείται από ενδογενή στοιχεία Τα αποτελέσματά μας αποδεικνύουν για πρώτη φορά ότι ο ενδογενής ρετροϊός HERV-K10 είναι ρετρομεταθετικά ενεργός, ακόμα και ελλειμματικός, και ότι τα καρκινικά και αναπτυσσόμενα γαμετικά κύτταρα αποτελούν ευνοϊκό περιβάλλον για τη γένεση γεγονότων ρετρομετάθεσης. Έπειτα, μελετήσαμε το πρότυπο μεθυλίωσης DNA του ρετροτρανσποζονίου HERV-K10 και των γονιδίων με αποτύπωση DLK1/MEG3, CDKN1C (p57KIP2) και IGF2/H19 σε χοριακές λάχνες από παιδιά που γεννήθηκαν μετά από IVF-PGD και βιοψίες όρχεων και σπέρμα υπογόνιμων ανδρών με MS-PCR. Στην ομάδα PGD, διαπιστώθηκε στατιστικά σημαντική μείωση της μεθυλίωσης του HERV-K10 και του DLK1/MEG3 σε σύγκριση με το μάρτυρα. Σε βιοψίες όρχεων βρέθηκε εκτεταμένη διαταραχή των προτύπων μεθυλίωσης γονιδίων με αποτύπωση. Στο σπέρμα διαπιστώθηκε ήπια διαταραχή της μεθυλίωσης των HERV-K10, ενώ η διαταραχή των γονιδίων με αποτύπωση που μελετήθηκαν ήταν κατά σειρά μειούμενης σφοδρότητας DLK1/MEG3>IGF2/H19>CDKN1C (p57KIP2). Η ανάλυση της αλληλουχίας των υπό μελέτη γονιδίων με εργαλεία βιοπληροφορικής κατέδειξε ότι το DLK1/MEG3 φέρει υψηλότατη περιεκτικότητα σε μεταθετά στοιχεία, το IGF2/H19 χαμηλή και το CDKN1C (p57KIP2) ακόμη χαμηλότερη. Σημαντική διαταραχή των προτύπων μεθυλίωσης γονιδίων με πατρικό αποτύπωμα φαίνεται να συσχετίζεται με σοβαρής μορφής υπογονιμότητα, ενώ η ταυτόχρονη διαταραχή των προτύπων μεθυλίωσης των γονιδίων DLK1/MEG3 και IGF2/H19 θα μπορούσαν πιθανώς να αποτελέσουν δείκτη της ποιότητας του σπέρματος. Φαίνεται ότι η μεταβολή των προτύπων μεθυλίωσης του HERV-K10 συσχετίζεται με εκείνη γονιδίων με αποτύπωση που βρίσκονται σε περιοχές πλούσιες σε μεταθετά στοιχεία

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

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

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Transfer of aneuploid embryos following preimplantation gegnetic diagnosis: the added value of a haplotypign-base genome-wide approach

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Reciprocal 22q11.2 Deletion and Duplication in Siblings with Karyotypically Normal Parents

The 22q11.2 locus is known to harbor a high risk for structural variation caused by non-allelic homologous recombination, resulting in deletions and duplications. Here, we describe the first family with one sibling carrying the 22q11 deletion and the other carrying the reciprocal duplication. FISH and SNP array analysis of the parents show a maternal origin for both deletion and duplication, without indications of balanced deletions/duplications or mosaicism. We hypothesize that germline mosaicism in the mother underlies the deletion and duplication, which would implicate a high recurrence risk for her offspring.status: publishe

Refining the locus of branchio-otic syndrome 2 (BOS2) to a 5.25 Mb locus on chromosome 1q31.3q32.1

In 1991, a large family was described with an autosomal dominant inheritance of otological and branchial manifestations which was termed branchio-otic syndrome type 2 (BOS2). This trait was mapped by linkage analysis in this family to a region of 23-31 Mb on chromosome 1q25.1q32.1. In the present report we describe the clinical features of two patients with a deletion in this region: one patient has a deletion but no otological or branchial manifestations, the other patient manifests mild conductive hearing loss resulting from bilaterally malformed middle ear ossicles, as well as a preauricular pit. Mapping of the deletion breakpoints allowed to delineate the region involved in BOS2 to a 5.25 Mb region containing 27 protein-coding genes. A detailed medical history of both patients is provided and they are compared with the literature on other detected interstitial deletions of 1q25q32. These findings will aid in the identification of the genetic cause underlying BOS2.status: publishe

ESHRE PGT Consortium good practice recommendations for the detection of structural and numerical chromosomal aberrations

The field of preimplantation genetic testing (PGT) is evolving fast, and best practice advice is essential for regulation and standardisation of diagnostic testing. The previous ESHRE guidelines on best practice for PGD, published in 2005 and 2011, are considered outdated, and the development of new papers outlining recommendations for good practice in PGT was necessary. The current paper provides recommendations on the technical aspects of PGT for chromosomal structural rearrangements (PGT-SR) and PGT for aneuploidies (PGT-A) and covers recommendations on array-based comparative genomic hybridisation (aCGH) and next-generation sequencing (NGS) for PGT-SR and PGT-A and on fluorescence in situ hybridisation (FISH) and single nucleotide polymorphism (SNP) array for PGT-SR, including laboratory issues, work practice controls, pre-examination validation, preclinical work-up, risk assessment and limitations. Furthermore, some general recommendations on PGT-SR/PGT-A are formulated around training and general risk assessment, and the examination and post-examination process. This paper is one of a series of four papers on good practice recommendations on PGT. The other papers cover the organisation of a PGT centre, embryo biopsy and tubing and the technical aspects of PGT for monogenic/single-gene defects (PGT-M). Together, these papers should assist everyone interested in PGT in developing the best laboratory and clinical practice possible