Browsing by Author "Kurg, Ants"

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A dual colour FISH method for routine validation of sexed Bos taurus semen
(BMC Veterinary Research, 2019) Reinsalu, Olavi; Mikelsaar, Ruth; Mikelsaar, Aavo-Valdur; Hallap, Triin; Jaakma, Ülle; Padrik, Peeter; Kavak, Ants; Salumets, Andres; Kurg, Ants
Background Usage of sexed semen that allows to choose the gender of the calves, is commonly practiced in livestock industry as a profitable breeding alternative, especially in dairy farming. The flow cytometric cell sorting is the only commercially available method for bovine sperm sexing. For validation of the sexing procedure several methods have been developed including sperm fluorescence in situ hybridisation techniques. Latter usually include the use of pre-labelled nucleotides for probe synthesis which is relatively expensive approach compared to combined application of aminoallyl-dUTP and chemical binding of fluorescent dyes. Here a sex determining dual colour bovine sperm fluorescence in situ hybridisation method is presented which is considered more cost-effective technique than the previously reported approaches. Results The reliability of sex chromosome identifying probes, designed in silico, was proven on bovine metaphase plate chromosomes and through comparison with a commercially available standard method. In the dual colour FISH experiments of unsexed and sexed bovine sperm samples the hybridisation efficiency was at least 98%, whereas the determined sex ratios were not statistically different from the expected. Very few cells carried both of the sex chromosome-specific signals (less than 0.2%). Conclusions A protocol for a dual colour bovine sperm FISH method is provided which is cost-effective, simple and fast for sex determination of spermatozoa in bull semen samples.
A speculative outlook on embryonic aneuploidy: Can molecular pathways be involved?
(2018) Tšuiko, Olga; Jatsenko, Tatjana; Parameswaran Grace, Lalit Kumar; Kurg, Ants; Vermeesch, Joris Robert; Lanner, Fredrik; Altmäe, Signe; Salumets, Andres
The journey of embryonic development starts at oocyte fertilization, which triggers a complex cascade of events and cellular pathways that guide early embryogenesis. Recent technological advances have greatly expanded our knowledge of cleavage-stage embryo development, which is characterized by an increased rate of whole-chromosome losses and gains, mixoploidy, and atypical cleavage morphokinetics. Embryonic aneuploidy significantly contributes to implantation failure, spontaneous miscarriage, stillbirth or congenital birth defects in both natural and assisted human reproduction. Essentially, early embryo development is strongly determined by maternal factors. Owing to considerable limitations associated with human oocyte and embryo research, the use of animal models is inevitable. However, cellular and molecular mechanisms driving the error-prone early stages of development are still poorly described. In this review, we describe known events that lead to aneuploidy in mammalian oocytes and preimplantation embryos. As the processes of oocyte and embryo development are rigorously regulated by multiple signal-transduction pathways, we explore the putative role of signaling pathways in genomic integrity maintenance. Based on the existing evidence from human and animal data, we investigate whether critical early developmental pathways, like Wnt, Hippo and MAPK, together with distinct DNA damage response and DNA repair pathways can be associated with embryo genomic instability, a question that has, so far, remained largely unexplored.
Bifunktsionaalsed streptavidiini teisendid: disain ja ekspressioon E. coli’s
(Tartu Ülikool, 2016) Pedor, Jenni Katri; Kurg, Ants; Kolmer, Meelis; Tartu Ülikool. Loodus- ja tehnoloogiateaduskond; Tartu Ülikool. Molekulaar- ja rakubioloogia instituut
Käesolevas bakalaureusetöös disainiti ja toodeti rekombinatseid valke, mis võimaldavad kinnitada märgistatud bioloogilisi märklaudmolekule nii raku membraani kui tuuma ja tsütoplasmasse. Märklaudmolekulide sidumiseks kasutati streptavidiini teisendeid: tetrameere moodustavat streptavidiini ja kimäärset monomeerset streptavidiini. Raku membraani külge kinnituvaks domeeniks valiti bakteriorodopsiini modifitseeritud C-heeliks ning rekombinantse valgu lokaliseerimiseks raku tuuma ja tsütoplasmasse valiti penetratiin. E. coli’s ekspresseerusid C-heeliksist ja kimäärsest monomeersest streptavidiinist koosnev rekombinantne valk ning penetratiinist ja streptavidiinist koosnev rekombinantne valk. Penetratiinist ja streptavidiinist koosnev rekombinantne valk moodustas tetrameere ja sidus biotiini.
Creating basis for introducing non‐invasive prenatal testing in the Estonian public health setting
(John Wiley & Sons, Ltd., 2019-11) Žilina, Olga; Rekker, Kadri; Kaplinski, Lauris; Sauk, Martin; Paluoja, Priit; Teder, Hindrek; Ustav, Eva‐Liina; Tõnisson, Neeme; Reimand, Tiia; Ridnõi, Konstantin; Palta, Priit; Vermeesch, Joris Robert; Krjutškov, Kaarel; Kurg, Ants; Salumet, Andres
Objective The study aimed to validate a whole‐genome sequencing‐based NIPT laboratory method and our recently developed NIPTmer aneuploidy detection software with the potential to integrate the pipeline into prenatal clinical care in Estonia. Method In total, 424 maternal blood samples were included. Analysis pipeline involved cell‐free DNA extraction, library preparation and massively parallel sequencing on Illumina platform. Aneuploidies were determined with NIPTmer software, which is based on counting pre‐defined per‐chromosome sets of unique k‐mers from sequencing raw data. SeqFF was implemented to estimate cell‐free fetal DNA (cffDNA) fraction. Results NIPTmer identified correctly all samples of non‐mosaic trisomy 21 (T21, 15/15), T18 (9/9), T13 (4/4) and monosomy X (4/4) cases, with the 100% sensitivity. However, one mosaic T18 remained undetected. Six false‐positive (FP) results were observed (FP rate of 1.5%, 6/398), including three for T18 (specificity 99.3%) and three for T13 (specificity 99.3%). The level of cffDNA of <4% was estimated in eight samples, including one sample with T13 and T18. Despite low cffDNA level, these two samples were determined as aneuploid. Conclusion We believe that the developed NIPT method can successfully be used as a universal primary screening test in combination with ultrasound scan for the first trimester fetal examination.
Genome stability of bovine in vivo-conceived cleavage-stage embryos is higher compared to in vitro-produced embryos.
(2017) Tšuiko, Olga; Catteeuw, Maaike; Zamani Esteki, Masoud; Destouni, Aspasia; Bogado Pascottini, Osvaldo; Besenfelder, Urban; Havlicek, Vitezslav; Smits, Katrien; Kurg, Ants; Salumets, Andres; D’Hooghe, Thomas; Voet, Thierry; Van Soom, Ann; Vermeesch, Joris Robert
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)
In vitro fertilization does not increase the incidence of de novo copy number alterations in fetal and placental lineages
(Nature Medicine, 2019-11-04) Esteki, Masoud Zamani; Viltrop, Triin; Tšuiko, Olga; Tiirats, Airi; Koel, Mariann; Nõukas, Margit; Žilina, Olga; Teearu, Katre; Marjonen, Heidi; Kahila, Hanna; Meekels, Jeroen; Söderström-Anttila, Viveca; Suikkari, Anne-Maria; Tiitinen, Aila; Mägi, Reedik; Kõks, Sulev; Kaminen-Ahola, Nina; Kurg, Ants; Voet, Thierry; Vermeesch, Joris Robert; Salumets, Andres
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.
Karyotype of the blastocoel fluid demonstrates low concordance with both trophectoderm and inner cell mass
(2018-06) Tšuiko, Olga; Zhigalina, Daria I.; Jatsenko, Tatjana; Skryabin, Nikolay A.; Kanbekova, Olga R.; Artyukhova, Victoria G.; Svetlakov, Anatoly V.; Teearu, Katre; Trošin, Aleksander; Salumets, Andres; Kurg, Ants; Lebedev, Igor N.
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.
Liquid Biopsy Approach in Precision Oncology
(2020) Ruiz Acosta, Alberto; Kurg, Ants
Liquid biopsy serves as a tool to analyse cell-free nucleic acids (cfNAs) and circulating tu-mour cells (CTCs) in blood or other body fluids in individuals with cancer. These are con-sidered promising diagnostic and prognostic biomarkers but new discoveries are still being made in regard to their heterogeneity, therefore it is critical to continue researching to demonstrate a clinical advantage in precision oncology. For now they promise to reveal mo-lecular characterisation of tumours which can change the prognosis, monitoring and post-therapy of cancer care. This is challenging because the technologies and methodologies are still not mature enough for capturing and isolating viable cells. Higher affinity and sensitive techniques including a combination of these may result in an efficient characterisation of CTCs and cfNAs. From pre-treatment of blood samples to post-analysis of isolated markers it is required to standardize and regulate methods in order to prove clinical utility, as of now this remains in the experimental phase. Microfluidic systems have become an influential tool in cancer studies for their capability to analyse single cells or molecules and its integration with other techniques, miniaturization and fast analysis which will help overcome challenges these biomarkers face to eventually replace traditional biopsy with liquid biopsy. In estonian: Bakalaureusetöö on kirjutatud inglise keeles, kirjanduse põhjal ning on klassikalise ülesehi-tusega koosnedes sissejuhatusest, kirjanduse ülevaatest, töö eesmärkidest, diskussioonist ja kokkuvõttest. Töö on esitatud 40-l leheküljel, sisaldades ka ühe joonise ja neli tabelit. Töö koostamisel on kasutatud 48 kirjanduse allikat. Töö põhisisuks on ülevaate andmine erinevatest vedelbiopsia meetoditest, mille abil saab vähi patsientidel analüüsida veres või muudes kehavedelikes ringlevaid rakuvabasid nuk-leiinhappeid (cfNAd) ja ringlevaid vähirakke (CTCd). Neid peetakse paljutõotavateks diag-nostilisteks ja prognostilisteks biomarkeriteks, mis võimaldavad senisest paremini ning mitte-invasiivselt iseloomustada kasvajaid molekulaarsel tasemel. See on väga oluline vähi patsientide personaalses ravis, võimaldades kasvajate tekke ja arengu paremat jälgimist ning ravi. Töös on kirjeldatud cfNA-de ja CTC-de olemust, päritolu, eraldamist ning analüüsiks kasutatavaid tehnoloogiaid. Samuti on võrreldud vedelbiopsia kasutatavust ja perspektiive tänapäeval traditsiooniliselt rakendatava klassikalise biopsia meetoditega. Arutelu osas on antud ülevaade meetodite hetkeseisust ja võimalikest arengutest tulevikus.
Utilising FGF2, IGF2 and FSH in serum-free protocol for long-term in vitro cultivation of primary human granulosa cells
(2020) Hensen, Kati; Pook, Martin; Sikut, Anu; Org, Tõnis; Maimets, Toivo; Salumets, Andres; Kurg, Ants
Human granulosa cells acquired as leftover from IVF treatment can be used to study infertility problems and area valuable tool in the research of follicle maturation and ovulation. There is a need for more defined and long-term culture protocols for studying the response of granulosa cells upon treatment with selected hormones/chemicals. In the current study, we tested the effect of adding FGF2, IGF2 and FSH into defined basal medium inorder tofind culture conditions that would support proliferation of cumulus and mural granulosa cells alongwith the expression of common granulosa cell type markers such asFSHR,AMHR2,LHRandCYP19A1. We foundthat FGF2, IGF2 together with FSH helped to retain granulosa cell marker expression while supporting cellsurvival at least for two weeks of culture. The defined serum-free culture conditions for long-term culturing willbe valuable in providing new standards in the research of human granulosa cells.