WIDENLIFE - Widening the Scientific Excellence for Studies on Women’s and Fetal Health and Wellbeing

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Given that one in six couples face infertility, often caused by female factors, women’s reproductive health is a significant medical and socio-economical challenge. Herein, while addressing this issue, the Estonian research on female reproductive health and medicine has rapidly developed with the lead of University of Tartu (UT). Nevertheless, the UT’s capacity and expertise still falls short of the leading institutions. At the same time, also the most competitive research groups face significant barriers to perform world-class science due to substantial networking gaps that still exist between previously non-linked research teams. TWINNING funding scheme is designed to overcome these aforementioned shortcomings. Thus UT has formed a WIDENLIFE consortium with its world-renowned partners: University of Oxford and Katholieke Universiteit Leuven, and altogether we have set two ambitious objectives to address. Firstly, for the UT: to become one of the leading research and teaching centres for reproductive and fetal medicine in Eastern and Northern Europe. Secondly, for all members of consortia: to intensify trilateral synergies between the research groups in the areas of female reproductive health and medicine. Our specific goals are to highlight the associations between female metabolic health and infertility, provide deeper understating for embryonal development, and offer new tools for infertility treatment and prenatal diagnostics. In order to resolve these objectives, exchange of know-how, ideas and information between the partners will be enhanced, creating the novel clinically valuable information through pooling the expertise and synergy of resources, interests and commitments by universities from Estonia, UK and Belgium. This could also mean a significant contribution to the scientific capacity of the Estonian research community as well as the health technology industry, which is one of the main focus areas for Estonian Smart Specialisation Strategy.


Recent Submissions

Now showing 1 - 20 of 45
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    The genetic architecture of sporadic and multiple consecutive miscarriage
    (Nature Communications, 2020-11-25) Laisk, Triin; Soares, Ana Luiza G.; Lindgren, Cecilia M.; Ferreira, Teresa; Painter, Jodie N.; Censin, Jenny C.; Laber, Samantha; Bacelis, Jonas; Chen, Chia-Yen; Lepamets, Maarja; Lawlor, Deborah A.; Mägi, Reedik; Medland, Sarah E.; Granne, Ingrid; Walters, Robin G.; Nielsen, Rasmus; Neale, Benjamin M.; Martin, Nicholas G.; Li, Liming; Jacobsson, Bo; Conrad, Donald F.; Chen, Zhengming; Werge, Thomas; Zondervan, Krina; Snieder, Harold; Salumets, Andres; Seminara, Stephanie; Lippincott, Margaret; Nyholt, Dale R.; Nordentoft, Merete; Njølstad, Pål R.; Mortensen, Preben B.; Mors, Ole; Morris, Andrew P.; Montgomery, Grant W.; Metspalu, Andres; Lind, Penelope A.; Kukushkina, Viktorija; Kartsonaki, Christiana; Juodakis, Julius; Johansson, Stefan; Jin, Xin; Hougaard, David M.; Helgeland, Øyvind; Bybjerg-Grauholm, Jonas; Gordon, Scott D.; Børglum, Anders D.; Becker, Christian M.; Yang, Ling; Andersen, Marianne S.; Southcombe, Jennifer; Ramu, Avinash; Millwood, Iona Y.; Liu, Siyang; Lin, Kuang
    Miscarriage is a common, complex trait affecting ~15% of clinically confirmed pregnancies. Here we present the results of large-scale genetic association analyses with 69,054 cases from five different ancestries for sporadic miscarriage, 750 cases of European ancestry for multiple (≥3) consecutive miscarriage, and up to 359,469 female controls. We identify one genome-wide significant association (rs146350366, minor allele frequency (MAF) 1.2%, P = 3.2 × 10−8, odds ratio (OR) = 1.4) for sporadic miscarriage in our European ancestry meta-analysis and three genome-wide significant associations for multiple consecutive miscarriage (rs7859844, MAF = 6.4%, P = 1.3 × 10−8, OR = 1.7; rs143445068, MAF = 0.8%, P = 5.2 × 10−9, OR = 3.4; rs183453668, MAF = 0.5%, P = 2.8 × 10−8, OR = 3.8). We further investigate the genetic architecture of miscarriage with biobank-scale Mendelian randomization, heritability, and genetic correlation analyses. Our results show that miscarriage etiopathogenesis is partly driven by genetic variation potentially related to placental biology, and illustrate the utility of large-scale biobank data for understanding this pregnancy complication.
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    Understanding the mechanisms of endometrial receptivity through integration of ‘omics’ data layers
    (2020-05-28) Kukuškina, Viktorija; Mägi, Reedik, juhendaja; Metspalu, Andres, juhendaja; Salumets, Andres, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond
    Maailma Terviseorganisatsiooni statistika väidab, et umbes 10% püsisuhetes olevatest naistest on ühel või teisel põhjusel viljatud. Naise viljakust mõjutab välja palju erinevaid faktoreid ning setõttu on viljatuse põhjuste leidmine tihti väga keeruline. Viljatust põhjustavateks faktoriteks võivad olla üldine terviseseisund, erinevad haigused, geneetiline taust, väliskeskkonna ja eluviisiga seotud tegurid. Ühe näitena võib tuua embrüo pesastumist (implantatsioon) emaka limaskesta (endomeetriumi), mis võib toimuda ainult kindla lühikese perioodi vältel (implantatsiooni aken), kui endomeetrium on embrüo suhtes kõige vastuvõtlikum. Implantatsiooni akna periood on aga iga naise jaoks erinev, ning on määratud erinevate bioloogiliste protsesside poolt. Kunstliku viljastamise (IVF) läbiviimise jaoks on kriitiline teada täpset implantatsiooni akna aega, sellega seotud mehhanisme ja nende vastastikust mõju. Selleks, et uurida mehhanismide omavahelisi seoseid, panime paariviisiliselt kokku erinevaid geneetilise regulatsiooni andmekihte, milleks olid RNA, mikroRNA ja DNA metülatsiooni admed, ja mida koos nimetatakse ‘oomika’ andmekihtideks. Kokkuvõtvalt näitavad antud töö tulemused, et, võrreldes ühe ‘oomika’ andmekihi uurimisega, ‘oomika’ andmekihtide kombineerimine aitab paremini mõista endomeetriumi retseptiivsusega seotud bioloogilisi protsesse ning vältida valepositiivseid tulemusi. Antud tööga me rõhutame süsteemibioloogia ning paljude andmekihtide samaaegse kasutamise olulisust naise reproduktiivsuse bioloogiliste mehhanismide uurimisel.
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    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.
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    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.
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    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.
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    A molecular tool for menstrual cycle phase dating of endometrial samples in endometriosis transcriptome studies
    (2019) Saare, Merli; Merli, Triin; Teder, Hindrek; Paluoja, Priit; Palta, Priit; Koel, Mariann; Kirss, Fred; Karro, Helle; Sõritsa, Deniss; Salumets, Andres; Krjutškov, Kaarel; Peters, Maire
    Transcriptome profiling of 57 endometrial receptivity genes specifies the menstrual cycle phase of endometrial samples.
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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.
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    Ranking and characterization of established BMI and lipid associated loci as candidates for gene-environment interactions
    (2017) Shungin, Dmitry; Deng, Wei Q.; Varga, Tibor V.; Luan, Jian'an; Mihailov, Evelin; Metspalu, Andres; GIANT Consortium; Morris, Andrew P.; Forouhi, Nita G.; Lindgren, Cecilia; Magnusson, Patrik K. E.; Pedersen, Nancy L.; Hallmans, Göran; Chu, Audrey Y.; Justice, Anne E.; Graff, Mariaelisa; Winkler, Thomas W.; Rose, Lynda M.; Langenberg, Claudia; Cupples, L. Adrienne; Kilpeläinen, Tuomas O.; Scott, Robert A.; Mägi, Reedik; Paré, Guillaume; Franks, Paul W.; Ridker, Paul M.; Wareham, Nicholas J.; Ong, Ken K.; Loos, Ruth J. F.; Chasman, Daniel I.; Ingelsson, Erik
    Phenotypic variance heterogeneity across genotypes at a single nucleotide polymorphism (SNP) may reflect underlying gene-environment (G×E) or gene-gene interactions. We modeled variance heterogeneity for blood lipids and BMI in up to 44,211 participants and investigated relationships between variance effects (Pv), G×E interaction effects (with smoking and physical activity), and marginal genetic effects (Pm). Correlations between Pv and Pm were stronger for SNPs with established marginal effects (Spearman’s ρ = 0.401 for triglycerides, and ρ = 0.236 for BMI) compared to all SNPs. When Pv and Pm were compared for all pruned SNPs, only BMI was statistically significant (Spearman’s ρ = 0.010). Overall, SNPs with established marginal effects were overrepresented in the nominally significant part of the Pv distribution (Pbinomial <0.05). SNPs from the top 1% of the Pm distribution for BMI had more significant Pv values (PMann–Whitney = 1.46×10−5), and the odds ratio of SNPs with nominally significant (<0.05) Pm and Pv was 1.33 (95% CI: 1.12, 1.57) for BMI. Moreover, BMI SNPs with nominally significant G×E interaction P-values (Pint<0.05) were enriched with nominally significant Pv values (Pbinomial = 8.63×10−9 and 8.52×10−7 for SNP × smoking and SNP × physical activity, respectively). We conclude that some loci with strong marginal effects may be good candidates for G×E, and variance-based prioritization can be used to identify them.
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    The Influence of Different Maternal Microbial Communities on the Development of Infant Gut and Oral Microbiota
    (2017) Drell, Tiina; Štšepetova, Jelena; Simm, Jaak; Rull, Kristiina; Aleksejeva, Aira; Antson, Anne; Tillmann, Vallo; Metsis, Madis; Sepp, Epp; Salumets, Andres; Mändar, Reet
    Very few studies have analyzed how the composition of mother’s microbiota affects the development of infant’s gut and oral microbiota during the first months of life. Here, microbiota present in the mothers’ gut, vagina, breast milk, oral cavity, and mammary areola were compared with the gut and oral microbiota of their infants over the first six months following birth. Samples were collected from the aforementioned body sites from seven mothers and nine infants at three different time points over a 6-month period. Each sample was analyzed with 16S rRNA gene sequencing. The gut microbiota of the infants harbored distinct microbial communities that had low similarity with the various maternal microbiota communities. In contrast, the oral microbiota of the infants exhibited high similarity with the microbiota of the mothers’ breast milk, mammary areola and mouth. These results demonstrate that constant contact between microbial communities increases their similarity. A majority of the operational taxonomic units in infant gut and oral microbiota were also shared with the mothers’ gut and oral communities, respectively. The disparity between the similarity and the proportion of the OTUs shared between infants’ and mothers’ gut microbiota might be related to lower diversity and therefore competition in infants’ gut microbiota.
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    The management of menopause in women with a history of endometriosis: a systematic review
    (2017) Gemmell, L.C.; Webster, K.E.; Kirtley, S.; Vincent, K.; Zondervan, K.T.; Becker, C.M.
    BACKGROUND Endometriosis is typically regarded as a premenopausal disease, resolving after natural or iatrogenic menopause due to declining oestrogen levels. Nonetheless, case reports over the years have highlighted the incidence of recurrent postmenopausal endometriosis. It is now clear that both recurrence and malignant transformation of endometriotic foci can occur in the postmenopausal period. Postmenopausal women are commonly treated with hormone replacement therapy (HRT) to treat climacteric symptoms and prevent bone loss; however, HRT may reactivate endometriosis and stimulate malignant transformation in women with a history of endometriosis. Given the uncertain risks of initiating HRT, it is difficult to determine the best menopausal management for this group of women. OBJECTIVE AND RATIONAL The aim of this study was to systematically review the existing literature on management of menopausal symptoms in women with a history of endometriosis. We also aimed to evaluate the published literature on the risks associated with HRT in these women, and details regarding optimal formulations and timing (i.e. initiation and duration) of HRT. SEARCH METHODS Four electronic databases (MEDLINE via OVID, Embase via OVID, PsycINFO via OVID and CINAHL via EbscoHost) were searched from database inception until June 2016, using a combination of relevant controlled vocabulary terms and free-text terms related to ‘menopause’ and ‘endometriosis’. Inclusion criteria were: menopausal women with a history of endometriosis and menopausal treatment including HRT or other preparations. Case reports/series, observational studies and clinical trials were included. Narrative review articles, organizational guidelines and conference abstracts were excluded, as were studies that did not report on any form of menopausal management. Articles were assessed for risk of bias and quality using GRADE criteria. OUTCOMES We present a synthesis of the existing case reports of endometriosis recurrence or malignant transformation in women undergoing treatment for menopausal symptoms. We highlight common presenting symptoms, potential risk factors and outcomes amongst the studies. Sparse high-quality evidence was identified, with few observational studies and only two randomized controlled trials. Given this paucity of data, no definitive conclusions can be drawn concerning risk. WIDER IMPLICATIONS Due to the lack of high-quality studies, it remains unclear how to advise women with a history of endometriosis regarding the management of menopausal symptoms. The absolute risk of disease recurrence and malignant transformation cannot be quantified, and the impact of HRT use on these outcomes is not known. Multicentre randomized trials or large observational studies are urgently needed to inform clinicians and patients alike.
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    Large-scale meta-analysis highlights the hypothalamic–pituitary–gonadal axis in the genetic regulation of menstrual cycle length
    (Human Molecular Genetics, 2018) Laisk, Triin; Kukuškina, Viktorija; Palmer, Duncan; Laber, Samantha; Chen, Chia-Yen; Ferreira, Teresa; Rahmioglu, Nilufer; Zondervan, Krina; Becker, Christian; Smoller, Jordan W; Lippincott, Margaret; Salumets, Andres; Granne, Ingrid; Seminara, Stephanie; Neale, Benjamin; Mägi, Reedik; Lindgren, Cecilia M
    The normal menstrual cycle requires a delicate interplay between the hypothalamus, pituitary and ovary. Therefore, its length is an important indicator of female reproductive health. Menstrual cycle length has been shown to be partially controlled by genetic factors, especially in the follicle-stimulating hormone beta-subunit (FSHB) locus. A genome-wide association study meta-analysis of menstrual cycle length in 44 871 women of European ancestry confirmed the previously observed association with the FSHB locus and identified four additional novel signals in, or near, the GNRH1, PGR, NR5A2 and INS-IGF2 genes. These findings not only confirm the role of the hypothalamic–pituitary–gonadal axis in the genetic regulation of menstrual cycle length but also highlight potential novel local regulatory mechanisms, such as those mediated by IGF2.
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    GWAS Identifies Risk Locus for Erectile Dysfunction and Implicates Hypothalamic Neurobiology and Diabetes in Etiology
    (2019) Bovijn, J; Jackson, L; Censin, J; Chen, CY; Laisk, T; Laber, S; Ferreira, T; Pulit, SL; Glastonbury, CA; Smoller, JW; Harrison, JW; Ruth, KS; Beaumont, RN; Jones, SE; Tyrrell, J; Wood, AR; Weedon, MN; Mägi, R; Neale, B; Lindgren, CM; Murray, A; Holmes, MV
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    Genome-wide haplotyping embryos developing from 0PN and 1PN zygotes increases transferrable embryos in PGT-M.
    (2018) Destouni, Aspasia; Dimitriadou, Eftychia; Masset, Heleen; Debrock, Sophie; Melotte, Cindy; Bogaert, Kris Van Den; Esteki, Masoud Zamani; Ding, Jia; Voet, Thiery; Denayer, Ellen; de Ravel, Thomy; Legius, Eric; Meuleman, Christel; Peeraer, Karen; Vermeesch, Joris R
    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.
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    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.
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    Large-scale genome-wide meta-analysis of polycystic ovary syndrome suggests shared genetic architecture for different diagnosis criteria
    (2018) Day, Felix; Karaderi, Tugce; Jones, Michelle R; Meun, Cindy; He, Chunyan; Drong, Alex; Kraft, Peter; Lin, Nan; Huang, Hongyan; Broer, Linda; Magi, Reedik; Saxena, Richa; Laisk-Podar, Triin; Urbanek, Margrit; Welt, Corrine K; Lindgren, Cecilia M; Franks, Steve; Thorsteinsdottir, Unnur; Stefansson, Kari; Ong, Ken; Anttila, Verneri; Neale, Benjamin M; Jarvelin, Marjo-Riitta; Hayes, Geoffrey M; Thorleifsson, Gudmar; Fernandez-Tajes, Juan; Mahajan, Anubha; Mullin, Benjamin H; Stuckey, Bronwyn G.A; Spector, Timothy D.; Wilson, Scott G; Goodarzi, Mark O; Davis, Lea; Obermeyer-Pietsch, Barbara; Uitterlinden, Andre G; Fauser, Bart; Kowalska, Irina; Visser, Jenny A; Anderson, Marianne; McCarthy, Mark I; Morin-Papunen, Laure; Salumets, Andres; Stener-Victorin, Elisabet; Ehrmann, David; Legro, Richard S; Styrkarsdottir, Unnur; Perry, John; Dunaif, Andrea; Laven, Joop S.E.
    Polycystic ovary syndrome (PCOS) is a disorder characterized by hyperandrogenism, ovulatory dysfunction and polycystic ovarian morphology. Affected women frequently have metabolic disturbances including insulin resistance and dysregulation of glucose homeostasis. PCOS is diagnosed with two different sets of diagnostic criteria, resulting in a phenotypic spectrum of PCOS cases. The genetic similarities between cases diagnosed based on the two criteria have been largely unknown. Previous studies in Chinese and European subjects have identified 16 loci associated with risk of PCOS. We report a fixed-effect, inverse-weighted-variance meta-analysis from 10,074 PCOS cases and 103,164 controls of European ancestry and characterisation of PCOS related traits. We identified 3 novel loci (near PLGRKT, ZBTB16 and MAPRE1), and provide replication of 11 previously reported loci. Only one locus differed significantly in its association by diagnostic criteria; otherwise the genetic architecture was similar between PCOS diagnosed by self-report and PCOS diagnosed by NIH or non-NIH Rotterdam criteria across common variants at 13 loci. Identified variants were associated with hyperandrogenism, gonadotropin regulation and testosterone levels in affected women. Linkage disequilibrium score regression analysis revealed genetic correlations with obesity, fasting insulin, type 2 diabetes, lipid levels and coronary artery disease, indicating shared genetic architecture between metabolic traits and PCOS. Mendelian randomization analyses suggested variants associated with body mass index, fasting insulin, menopause timing, depression and male-pattern balding play a causal role in PCOS. The data thus demonstrate 3 novel loci associated with PCOS and similar genetic architecture for all diagnostic criteria. The data also provide the first genetic evidence for a male phenotype for PCOS and a causal link to depression, a previously hypothesized comorbid disease. Thus, the genetics provide a comprehensive view of PCOS that encompasses multiple diagnostic criteria, gender, reproductive potential and mental health.
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    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.
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    Unravelling chromosomal instability in mammalian preimplantation embryos using single-cell genomics
    (Tartu : University of Tartu Press, 2018) Tšuiko, Olga; Salumets, Andres, juhendaja; Vermeesch, Joris Robert, juhendaja; Kurg, Ants, juhendaja
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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.
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    Endometrial receptivity revisited: endometrial transcriptome adjusted for tissue cellular heterogeneity
    (2018) Suhorutshenko, Marina; Kukushkina, Viktorija; Velthut-Meikas, Agne; Altmäe, Signe; Peters, Maire; Mägi, Reedik; Krjutškov, Kaarel; Koel, Mariann; Codoñer, Francisco M; Martinez-Blanch, Juan Fco; Vilella, Felipe; Simón, Carlos; Salumets, Andres; Laisk, Triin
    STUDY QUESTION Does cellular composition of the endometrial biopsy affect the gene expression profile of endometrial whole-tissue samples? SUMMARY ANSWER The differences in epithelial and stromal cell proportions in endometrial biopsies modify the whole-tissue gene expression profiles and affect the results of differential expression analyses. WHAT IS ALREADY KNOWN Each cell type has its unique gene expression profile. The proportions of epithelial and stromal cells vary in endometrial tissue during the menstrual cycle, along with individual and technical variation due to the method and tools used to obtain the tissue biopsy. STUDY DESIGN, SIZE, DURATION Using cell-population specific transcriptome data and computational deconvolution approach, we estimated the epithelial and stromal cell proportions in whole-tissue biopsies taken during early secretory and mid-secretory phases. The estimated cellular proportions were used as covariates in whole-tissue differential gene expression analysis. Endometrial transcriptomes before and after deconvolution were compared and analysed in biological context. PARTICIPANTS/MATERIAL, SETTING, METHODS Paired early- and mid-secretory endometrial biopsies were obtained from 35 healthy, regularly cycling, fertile volunteers, aged 23–36 years, and analysed by RNA sequencing. Differential gene expression analysis was performed using two approaches. In one of them, computational deconvolution was applied as an intermediate step to adjust for the proportions of epithelial and stromal cells in the endometrial biopsy. The results were then compared to conventional differential expression analysis. Ten paired endometrial samples were analysed with qPCR to validate the results. MAIN RESULTS AND THE ROLE OF CHANCE The estimated average proportions of stromal and epithelial cells in early secretory phase were 65% and 35%, and during mid-secretory phase, 46% and 54%, respectively, correlating well with the results of histological evaluation (r = 0.88, P = 1.1 × 10−6). Endometrial tissue transcriptomic analysis showed that approximately 26% of transcripts (n = 946) differentially expressed in receptive endometrium in cell-type unadjusted analysis also remain differentially expressed after adjustment for biopsy cellular composition. However, the other 74% (n = 2645) become statistically non-significant after adjustment for biopsy cellular composition, underlining the impact of tissue heterogeneity on differential expression analysis. The results suggest new mechanisms involved in endometrial maturation, involving genes like LINC01320, SLC8A1 and GGTA1P, described for the first time in context of endometrial receptivity. LARGE-SCALE DATA The RNA-seq data presented in this study is deposited in the Gene Expression Omnibus database with accession number GSE98386. LIMITATIONS REASONS FOR CAUTION Only dominant endometrial cell types were considered in gene expression profile deconvolution; however, other less frequent endometrial cell types also contribute to the whole-tissue gene expression profile. WIDER IMPLICATIONS OF THE FINDINGS The better understanding of molecular processes during transition from pre-receptive to receptive endometrium serves to improve the effectiveness and personalization of assisted reproduction protocols. Biopsy cellular composition should be taken into account in future endometrial ‘omics’ studies, where tissue heterogeneity could potentially influence the results. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by: Estonian Ministry of Education and Research (grant IUT34-16); Enterprise Estonia (EU48695); the EU-FP7 Eurostars program (NOTED, EU41564); the EU-FP7 Marie Curie Industry-Academia Partnerships and Pathways (SARM, EU324509); Horizon 2020 innovation program (WIDENLIFE, EU692065); MSCA-RISE-2015 project MOMENDO (No 691058) and the Miguel Servet Program Type I of Instituto de Salud Carlos III (CP13/00038); Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and European Regional Development Fund (FEDER): grants RYC-2016-21199 and ENDORE SAF2017-87526. Authors confirm no competing interests.
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    rs10732516 polymorphism at the IGF2/H19 locus associates with genotype-specific effects on placental DNA methylation and birth weight of newborns conceived by assisted reproductive technology
    (2018) Marjonen, Heidi; Auvinen, Pauliina; Kahila, Hanna; Tšuiko, Olga; Kõks, Sulev; Tiirats, Airi; Viltrop, Triin; Tuuri, Timo; Söderström-Anttila, Viveca; Suikkari, Anne-Maria; Salumets, Andres; Tiitinen, Aila; Kaminen-Ahola, Nina
    Background Assisted reproductive technology (ART) has been associated with low birth weight of fresh embryo transfer (FRESH) derived and increased birth weight of frozen embryo transfer (FET)-derived newborns. Owing to that, we focused on imprinted insulin-like growth factor 2 (IGF2)/H19 locus known to be important for normal growth. This locus is regulated by H19 imprinting control region (ICR) with seven binding sites for the methylation-sensitive zinc finger regulatory protein (CTCF). A polymorphism rs10732516 G/A in the sixth binding site for CTCF, associates with a genotype-specific trend to the DNA methylation. Due to this association, 62 couples with singleton pregnancies derived from FRESH (44 IVF/18 ICSI), 24 couples from FET (15 IVF/9 ICSI), and 157 couples with spontaneously conceived pregnancies as controls were recruited in Finland and Estonia for genotype-specific examination. DNA methylation levels at the H19 ICR, H19 DMR, and long interspersed nuclear elements in placental tissue were explored by MassARRAY EpiTYPER (n = 122). Allele-specific changes in the methylation level of H19 ICR in placental tissue (n = 26) and white blood cells (WBC, n = 8) were examined by bisulfite sequencing. Newborns’ (n = 243) anthropometrics was analyzed by using international growth standards. Results A consistent trend of genotype-specific decreased methylation level was observed in paternal allele of rs10732516 paternal A/maternal G genotype, but not in paternal G/maternal A genotype, at H19 ICR in ART placentas. This hypomethylation was not detected in WBCs. Also genotype-specific differences in FRESH-derived newborns’ birth weight and head circumference were observed (P = 0.04, P = 0.004, respectively): FRESH-derived newborns with G/G genotype were heavier (P = 0.04) and had larger head circumference (P = 0.002) compared to newborns with A/A genotype. Also, the placental weight and birth weight of controls, FRESH- and FET-derived newborns differed significantly in rs10732516 A/A genotype (P = 0.024, P = 0.006, respectively): the placentas and newborns of FET-derived pregnancies were heavier compared to FRESH-derived pregnancies (P = 0.02, P = 0.004, respectively). Conclusions The observed DNA methylation changes together with the phenotypic findings suggest that rs10732516 polymorphism associates with the effects of ART in a parent-of-origin manner. Therefore, this polymorphism should be considered when the effects of environmental factors on embryonic development are studied.