Kromosomaalne ebastabiilsus imetajate varajastes embrüotes

Väitekirja elektrooniline versioon ei sisalda publikatsiooneViljatus on kogu maailmas kiiresti kasvavaks probleemiks ning iga kuues paar seisab silmitsi olukorraga, et soovitud rasedust ei teki. Esmasünnitajate keskmine vanus kasvab samuti, kuid naise vananedes tema viljakus langeb ning seetõttu pöördub tänapäeval aina rohkem paare viljatusravi poole, et leida oma probleemile lahendus. Kehaväline viljastamine (in vitro fertilization, IVF), mille korral toimub munarakkude viljastamine ning embrüote areng katseklaasis, on maailmas kõige sagedamini kasutatav lastetusravi meetod. Paraku, IVF embrüote sagedased kromosomaalsed aberratsioonid on kõige suurem väljakutse IVF ravil, sest see on üks peamisi raseduse varajase katkemise (kuni 75%) ja IVF ebaõnnestumise põhjuseid. Samas, embrüotega seotud teadustöö on piiratud eetiliste põhjuste tõttu, seega sõltuvad inimese embrüote uuringud suurel määral sobivatest loommudelitest. Kasutades uudset üksikraku kogu-genoomi analüüsi metoodikat, näidati antud doktoritöös, et ka veise IVF embrüotes esinevad väga sagedased kromosomaalsed aberratsioonid, mille tulemusena on suurem osa varajasi embrüoid mosaiiksed. Lisaks avastati, et embrüo on võimeline segregeerima ema- ja isapoolse genoomi eraldi rakuliinidesse, mis võib-olla aluseks eriploidsuse (mixoploidy) ja kimääride tekkele, kuid selle nähtuse esinemissagedus on hetkel teadmata. Kromosoomi aberratsioonid eksisteerivad samuti loomulikul teel saadud embrüotes, kuid munarakkude ja embrüote in vitro manipuleerimine suurendab genoomi ebastabiilsuse sagedust veise varajastes embrüotes, vähendades nende elujõulisust. Kui sama trend kehtib ka inimese embrüote puhul, peaks see julgustama teadus- ja meditsiinikogukonda mitte ainult parandama in vitro kultuuri tingimusi, vaid ka tõstma inimeste teadlikkust viljakuse ja IVF ravi osas. Selleks, et tuvastada embrüo aneuploidiat kasutatakse tänapäeval embrüote sõeluuringut, analüüsides kas kolmanda päeva või viienda päeva biopsiat. Viimasel ajal on aga hakatud pöörama suuremat tähelepanu ka blastotsööli vedeliku (blastotsüsti sees olev vedelik) analüüsile, kuid antud töö käigus selgus, et blastotsööli vedeliku analüüsi tulemused on vastuolulised ning seda meetodit ei saa hetkel kasutada embrüote genoomi analüüsis.Fertility issues have become a plague of modern society, as one in six couples will encounter problems to achieve clinical pregnancy. Nowadays, natural conception is often taken for granted, leading to an increased number of couples that turn to assisted reproduction to help conceive and ultimately give birth to a healthy baby. In vitro fertilization (IVF) and embryo transfer is the most commonly performed procedure in ART. However, the uprising era of single-cell research has highlighted the alarming fact that human IVF embryos have high prevalence of chromosomal instability (CIN), which represents one of the most serious challenges in IVF. At the same time, the use of in vitro fertilization (IVF) treatment provides a unique opportunity to study the fundamentals of preimplantation human development, from fertilization to blastocyst stage of development. At the same time, embryo-related research is ethically one of the most complex areas of reproductive science that greatly relies on the use of appropriate animal models. By using novel single-cell genomics approaches, the current thesis has demonstrated that the nature and frequency of genomic abnormalities in in vitro cleavage-stage embryos is highly conserved between cattle and human. Additionally, heterogoneic cell division was discovered, by which the zygote segregates entire parental genomes into separate blastomere lineages, giving rise to mixoploidy and chimaerism, although the frequency of this phenomenon and the fate of mixoploid embryos remain to be investigated. By using the established bovine model it was demonstrated that chromosomal abnormalities are also present in naturally conceived embryos, but in vitro procedures exacerbate CIN in early embryos, compromising their survival rate. If this is the same for human this should encourage scientific and medical communities not only to refine and improve in vitro culture conditions, but also raise awareness among men and women of reproductive age. To tackle the issues of embryonic aneuploidy, embryo chromosome screening is performed, by analysing either day 3 or day 5 biopsy. Recently, the use of blastocoel fluid (BF) biopsy was proposed, but the data presented here shows that BF-DNA is not suitable for diagnostic purposes, and day 5 biopsy remains the safest option with the most reliable results

Unravelling chromosomal instability in mammalian preimplantation embryos using single-cell genomics

https://www.ester.ee/record=b5163270*es

Karyotype of the blastocoel fluid demonstrates low concordance with both trophectoderm and inner cell mass

Objective To compare the genomic profiles of blastocoel fluid (BF), inner cell mass (ICM), and trophectoderm (TE) cells derived from the same blastocyst. Design Prospective study. Setting Academic and in vitro fertilization units. Patient(s) Sixteen donated cryopreserved embryos at blastocyst stage. Intervention(s) BF, TE, and ICM cells were retrieved from each blastocyst for chromosome analysis by means of next-generation sequencing (NGS). Main Outcome Measure(s) Aneuploidy screening and assessment of mosaicism in BF, TE and ICM samples with subsequent comparison of genomic profiles between the three blastocyst compartments. Result(s) Out of 16 blastocysts, 10 BF samples and 14 TE and ICM samples provided reliable NGS data for comprehensive chromosome analysis. Only 40.0% of BF-DNA karyotypes were fully concordant with TE or ICM, compared with 85.7% concordance between TE and ICM. In addition, BF-DNA was burdened with mosaic aneuploidies and the total number of affected chromosomes in BF was significantly higher compared with the TE and ICM. Conclusion(s) BF-DNA can be successfully amplified and subjected to NGS, but owing to increased discordance with ICM and TE, BF does not adequately represent the status of the rest of the embryo. To overcome biologic and technical challenges associated with BF sampling and processing, blastocentesis would require improvement in both laboratory protocols and aneuploidy calling algorithms. Therefore, TE biopsy remains the most effective way to predict embryonic karyotype, and the use of BF as a single source of DNA for preimplantation genetic screening is not yet advised

Detection of a balanced translocation carrier through trophectoderm biopsy analysis: a case report

Balanced translocation carriers are burdened with fertility issues due to improper chromosome segregation in gametes, resulting in either implantation failure, miscarriage or birth of a child with chromosomal disorders. At the same time, these individuals are typically healthy with no signs of developmental problems, hence they often are unaware of their condition. Yet, because of difficulties in conceiving, balanced translocation carriers often turn to assisted reproduction, some of whom may also undergo preimplantation genetic testing for aneuploidy (PGT-A) to improve the likelihood of achieving a successful pregnancy.Peer reviewe

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

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

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

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