Browsing by Author "Rekker, Kadri"

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A Two-Cohort RNA-seq Study Reveals Changes in Endometrial and Blood miRNome in Fertile and Infertile Women
(2018) Rekker, Kadri; Altmäe, Signe; Suhorutshenko, Marina; Peters, Maire; Martinez-Blanch, Juan F.; Codoñer, Francisco M.; Vilella, Felipe; Simón, Carlos; Salumets, Andres; Velthut-Meikas, Agne
The endometrium undergoes extensive changes to prepare for embryo implantation and microRNAs (miRNAs) have been described as playing a significant role in the regulation of endometrial receptivity. However, there is no consensus about the miRNAs involved in mid-secretory endometrial functions. We analysed the complete endometrial miRNome from early secretory (pre-receptive) and mid-secretory (receptive) phases from fertile women and from patients with recurrent implantation failure (RIF) to reveal differentially expressed (DE) miRNAs in the mid-secretory endometrium. Furthermore, we investigated whether the overall changes during early to mid-secretory phase transition and with RIF condition could be reflected in blood miRNA profiles. In total, 116 endometrial and 114 matched blood samples collected from two different population cohorts were subjected to small RNA sequencing. Among fertile women, 91 DE miRNAs were identified in the mid-secretory vs. early secretory endometrium, while no differences were found in the corresponding blood samples. The comparison of mid-secretory phase samples between fertile and infertile women revealed 21 DE miRNAs from the endometrium and one from blood samples. Among discovered novel miRNAs, chr2_4401 was validated and showed up-regulation in the mid-secretory endometrium. Besides novel findings, we confirmed the involvement of miR-30 and miR-200 family members in mid-secretory endometrial functions.
Challenges in endometriosis miRNA studies - From tissue heterogeneity to disease specific miRNAs.
(2017) Saare, Merli; Rekker, Kadri; Laisk-Podar, Triin; Rahmioglu, Nilufer; Zondervan, Krina; Salumets, Andres; Götte, Martin; Peters, Maire
In order to uncover miRNA changes in endometriosis pathogenesis, both endometriotic lesions and endometrial biopsies, as well as stromal and epithelial cells isolated from these tissues have been investigated and a large number of dysregulated miRNAs have been reported. However, the concordance between the result of different studies has remained small. One potential explanation for limited overlap between the proposed disease-related miRNAs could be the heterogeneity in tissue composition, as some studies have compared highly heterogeneous whole-lesion biopsies with endometrial tissue, some have compared the endometrium from patients and controls, and some have used pure cell fractions isolated from lesions and endometrium. This review focuses on the results of published miRNA studies in endometriosis to reveal the potential impact of tissue heterogeneity on the discovery of disease-specific miRNA alterations in endometriosis. Additionally, functional studies that explore the roles of endometriosis-involved miRNAs are discussed.
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.
Differentially-Expressed miRNAs in Ectopic Stromal Cells Contribute to Endometriosis Development: The Plausible Role of miR-139-5p and miR-375
(2018) Rekker, Kadri; Tasa, Tõnis; Saare, Merli; Samuel, Külli; Kadastik, Ülle; Karro, Helle; Götte, Martin; Salumets, Andres; Peters, Maire
microRNA (miRNA) expression level alterations between endometrial tissue and endometriotic lesions indicate their involvement in endometriosis pathogenesis. However, as both endometrium and endometriotic lesions consist of different cell types in various proportions, it is not clear which cells contribute to variability in miRNA levels and the overall knowledge about cell-type specific miRNA expression in ectopic cells is scarce. Therefore, we utilized fluorescence-activated cell sorting to isolate endometrial stromal cells from paired endometrial and endometrioma biopsies and combined it with high-throughput sequencing to determine miRNA alterations in endometriotic stroma. The analysis revealed 149 abnormally expressed miRNAs in endometriotic lesions, including extensive upregulation of miR-139-5p and downregulation of miR-375 compared to eutopic cells. miRNA transfection experiments in the endometrial stromal cell line ST-T1b showed that the overexpression of miR-139-5p resulted in the downregulation of homeobox A9 (HOXA9) and HOXA10 expression, whereas the endothelin 1 (EDN1) gene was regulated by miR-375. The results of this study provide further insights into the complex molecular mechanisms involved in endometriosis pathogenesis and demonstrate the necessity for cell-type-specific analysis of ectopic tissues to understand the interactions between different cell populations in disease onset and progression.
High-throughput mRNA sequencing of stromal cells from endometriomas and endometrium
(2017) Rekker, Kadri; Saare, Merli; Eriste, Elo; Tasa, Tõnis; Kukuškina, Viktorija; Roost, Anne Mari; Anderson, Kristi; Samuel, Kadri; Karro, Helle; Salumets, Andres; Peters, Maire
The aetiology of endometriosis is still unclear and to find mechanisms behind the disease development, it is important to study each cell type from endometrium and ectopic lesions independently. The objective of this study was to uncover complete mRNA profiles in uncultured stromal cells from paired samples of endometriomas and eutopic endometrium. High-throughput mRNA sequencing revealed over 1300 dysregulated genes in stromal cells from ectopic lesions, including several novel genes in the context of endometriosis. Functional annotation analysis of differentially expressed genes highlighted pathways related to cell adhesion, extracellular matrix–receptor interaction and complement and coagulation cascade. Most importantly, we found a simultaneous upregulation of complement system components and inhibitors, indicating major imbalances in complement regulation in ectopic stromal cells. We also performed in vitro experiments to evaluate the effect of endometriosis patients’ peritoneal fluid (PF) on complement system gene expression levels, but no significant impact of PF on C3, CD55 and CFH levels was observed. In conclusion, the use of isolated stromal cells enables to determine gene expression levels without the background interference of other cell types. In the future, a new standard design studying all cell types from endometriotic lesions separately should be applied to reveal novel mechanisms behind endometriosis pathogenesis.
The putative role of microRNAs in endometriosis pathogenesis and potential in diagnostics
(2019-07-04) Rekker, Kadri; Peters, Maire, juhendaja; Salumets, Andres, juhendaja; Karro, Helle, juhendaja; Tartu Ülikool. Meditsiiniteaduste valdkond
Endometrioos on sage günekoloogiline haigus, mille korral emaka limaskest ehk endomeetrium kasvab kolletena väljaspool emakaõõnt. Haigusega kaasnevad sageli tugevad kõhuvalud ja viljatus. Vaatamata ulatuslikele uuringutele on endiselt ebaselge, miks mõnel naisel endomeetriumi rakud vales asukohas püsima jäävad ja kasvama hakkavad. Probleemiks on ka endometrioosi diagnoosimise piiratud võimalused, mistõttu ainus viis haiguse lõplikuks kindlakstegemiseks on invasiivne operatsioon. Viimasel ajal on uute endometrioosi patogeneesi kandidaatmarkeritena pakutud välja mikroRNA-sid (miRNA-d). miRNA-d on lühikesed RNA molekulid, mis reguleerivad geeniekspressiooni nii normaalsete kui ka patoloogiliste seisundite, muu hulgas endometrioosi korral. miRNA-sid on võimalik tuvastada ka kehavedelikest, mis teeb neist potentsiaalsed biomarkeri kandidaadid haiguse diagnoosimiseks. Minu uuringute eesmärgiks oligi välja selgitada miRNA-de võimalikke rolle endometrioosi patogeneesis ja hinnata spetsiifiliste vereplasma miRNA-de sobivust endometrioosi biomarkeritena. Näitasime, et vereplasma miRNA-de tase ei ole naistel mõjutatud menstruaaltsüklist ja kindlad vereplasma miRNA-d võimaldavad eristada endometrioosiga naisi patsientidest, kellel haigust ei esine. Samas avastasime, et haigusest mittesõltuvad tegurid, nagu ööpäevarütm ja individuaalne varieeruvus mõjutavad miRNA tasemeid sellisel määral, et nende võimekus endometrioosi kliiniliste biomarkeritena on vähene. Lisaks võimaldas uuringu uudne lähenemine, kus kasutasime miRNA tasemete tuvastamiseks endometrioosi kolletes kindlat rakupopulatsiooni terve koe asemel, välja pakkuda võimalikke mehhanisme, mis viivad endometrioosiga kaasneva valu ja viljatuse tekkeni.
TAC-seq: targeted DNA and RNA sequencing for precise biomarker molecule counting
(2018) Teder, Hindrek; Koel, Mariann; Paluoja, Priit; Jatsenko, Tatjana; Rekker, Kadri; Laisk-Podar, Triin; Kukuškina, Viktorija; Velthut-Meikas, Agne; Fjodorova, Olga; Peters, Maire; Kere, Juha; Salumets, Andres; Palta, Priit; Krjutškov, Kaarel
Targeted next-generation sequencing (NGS) methods have become essential in medical research and diagnostics. In addition to NGS sensitivity and high-throughput capacity, precise biomolecule counting based on unique molecular identifier (UMI) has potential to increase biomolecule detection accuracy. Although UMIs are widely used in basic research its introduction to clinical assays is still in progress. Here, we present a robust and cost-effective TAC-seq (Targeted Allele Counting by sequencing) method that uses UMIs to estimate the original molecule counts of mRNAs, microRNAs, and cell-free DNA. We applied TAC-seq in three different clinical applications and compared the results with standard NGS. RNA samples extracted from human endometrial biopsies were analyzed using previously described 57 mRNA-based receptivity biomarkers and 49 selected microRNAs at different expression levels. Cell-free DNA aneuploidy testing was based on cell line (47,XX, +21) genomic DNA. TAC-seq mRNA profiling showed identical clustering results to transcriptome RNA sequencing, and microRNA detection demonstrated significant reduction in amplification bias, allowing to determine minor expression changes between different samples that remained undetermined by standard NGS. The mimicking experiment for cell-free DNA fetal aneuploidy analysis showed that TAC-seq can be applied to count highly fragmented DNA, detecting significant (p = 7.6 × 10−4) excess of chromosome 21 molecules at 10% fetal fraction level. Based on three proof-of-principle applications we demonstrate that TAC-seq is an accurate and highly potential biomarker profiling method for advanced medical research and diagnostics.
The influence of menstrual cycle and endometriosis on endometrial methylome
(Clin Epigenetics, 2016-01) Saare, Merli; Modhukur, Vijayachitra; Suhorutshenko, Marina; Rajashekar, Balaji; Rekker, Kadri; Sõritsa, Deniss; Karro, Helle; Soplepmann, Pille; Sõritsa, Andrei; Lindgren, Cecilia M; Rahmioglu, Nilufer; Drong, Alexander; Becker, Christian M; Zondervan, Krina T; Salumets, Andres; Peters, Maire
BACKGROUND: Alterations in endometrial DNA methylation profile have been proposed as one potential mechanism initiating the development of endometriosis. However, the normal endometrial methylome is influenced by the cyclic hormonal changes, and the menstrual cycle phase-dependent epigenetic signature should be considered when studying endometrial disorders. So far, no studies have been performed to evaluate the menstrual cycle influences and endometriosis-specific endometrial methylation pattern at the same time. RESULTS: Infinium HumanMethylation 450K BeadChip arrays were used to explore DNA methylation profiles of endometrial tissues from various menstrual cycle phases from 31 patients with endometriosis and 24 healthy women. The DNA methylation profile of patients and controls was highly similar and only 28 differentially methylated regions (DMRs) between patients and controls were found. However, the overall magnitude of the methylation differences between patients and controls was rather small (Δβ ranging from -0.01 to -0.16 and from 0.01 to 0.08, respectively, for hypo- and hypermethylated CpGs). Unsupervised hierarchical clustering of the methylation data divided endometrial samples based on the menstrual cycle phase rather than diseased/non-diseased status. Further analysis revealed a number of menstrual cycle phase-specific epigenetic changes with largest changes occurring during the late-secretory and menstrual phases when substantial rearrangements of endometrial tissue take place. Comparison of cycle phase- and endometriosis-specific methylation profile changes revealed that 13 out of 28 endometriosis-specific DMRs were present in both datasets. CONCLUSIONS: The results of our study accentuate the importance of considering normal cyclic epigenetic changes in studies investigating endometrium-related disease-specific methylation patterns.