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

Permanent URI for this collectionhttps://hdl.handle.net/10062/63400

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.

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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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    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 novel hypothesis for histone-to-protamine transition in Bos taurus spermatozoa
    (2016) Sillaste, Gerly; Kaplinski, Lauris; Meier, Riho; Jaakma, Ülle; Eriste, Elo; Salumets, Andres
    DNA compaction with protamines in sperm is essential for successful fertilization. However, a portion of sperm chromatin remains less tightly packed with histones, which genomic location and function remain unclear. We extracted and sequenced histone-associated DNA from sperm of nine ejaculates from three bulls. We found that the fraction of retained histones varied between samples, but the variance was similar between samples from the same and different individuals. The most conserved regions showed similar abundance across all samples, whereas in other regions, their presence correlated with the size of histone fraction. This may refer to gradual histone–protamine transition, where easily accessible genomic regions, followed by the less accessible regions are first substituted by protamines. Our results confirm those from previous studies that histones remain in repetitive genome elements, such as centromeres, and added new findings of histones in rRNA and SRP RNA gene clusters and indicated histone enrichment in some spermatogenesis-associated genes, but not in genes of early embryonic development. Our functional analysis revealed significant overrepresentation of cGMP-dependent protein kinase G (cGMP-PKG) pathway genes among histone-enriched genes. This pathway is known for its importance in pre-fertilization sperm events. In summary, a novel hypothesis for gradual histone-to-protamine transition in sperm maturation was proposed. We believe that histones may contribute structural information into early embryo by epigenetically modifying centromeric chromatin and other types of repetitive DNA. We also suggest that sperm histones are retained in genes needed for sperm development, maturation and fertilization, as these genes are transcriptionally active shortly prior to histone-to-protamine transition.
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    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.
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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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    Aberrant expression of genes associated with stemness and cancer in endometria and endometrioma in a subset of women with endometriosis
    (2018) Ponandai-Srinivasan, Sakthivignesh; Andersson, Karin L; Nister, Monica; Saare, Merli; Hassan, Halima A; Varghese, Suby J; Peters, Maire; Salumets, Andres; Gemzell-Danielsson, Kristina; Lalitkumar, Parameswaran Grace Luther
    STUDY QUESTION Is there molecular evidence for a link between endometriosis and endometriosis-associated ovarian cancers (EAOC)? STUDY ANSWER We identified aberrant gene expression signatures associated with malignant transformation in a small subgroup of women with ovarian endometriosis. WHAT IS KNOWN ALREADY Epidemiological studies have shown an increased risk of EAOC in women with ovarian endometriosis. However, the cellular and molecular changes leading to EAOC are largely unexplored. STUDY DESIGN, SIZE, DURATION CD73+CD90+CD105+ multipotent stem cells/progenitors (SC cohort) were isolated from endometrium (n = 18) and endometrioma (n = 11) of endometriosis patients as well as from the endometrium of healthy women (n = 14). Extensive phenotypic and functional analyses were performed in vitro on expanded multipotent stem cells/progenitors to confirm their altered characteristics. Aberrant gene signatures were also validated in paired-endometrium and -endometrioma tissue samples from another cohort (Tissue cohort, n = 19) of endometriosis patients. PARTICIPANTS/MATERIALS, SETTINGS, METHODS Paired-endometrial and -endometriotic biopsies were obtained from women with endometriosis (ASRM stage III–IV) undergoing laparoscopic surgery. Control endometria were obtained from healthy volunteers. Isolated CD73+CD90+CD105+ SC were evaluated for the presence of known endometrial surface markers, colony forming efficiency, multi-lineage differentiation, cell cycle distribution and 3D-spheroid formation capacity. Targeted RT-PCR arrays, along with hierarchical and multivariate clustering tools, were used to determine both intergroup and intragroup gene expression variability for stem cell and cancer-associated markers, in both SC+ and tissue cohorts. MAIN RESULTS AND THE ROLE OF CHANCE Isolated and expanded SC+ from both control and patient groups showed significantly higher surface expression of W5C5+, clonal expansion and 3D-spheroid formation capacity (P < 0.05) compared with SC−. The SC+ cells also undergo mesenchymal lineage differentiation, unlike SC−. Gene expression from paired-endometriosis samples showed significant downregulation of PTEN, ARID1A and TNFα (P < 0.05) in endometrioma compared with paired-endometrium SC+ samples. Hierarchical and multivariate clustering from both SC+ and tissue cohorts together identified 4 out of 30 endometrioma samples with aberrant expression of stem cell and cancer-associated genes, such as KIT, HIF2α and E-cadherin, altered expression ratio of ER-β/ER-α and downregulation of tumour suppressor genes (PTEN and ARID1A). Thus, we speculate that above changes may be potentially relevant to the development of EAOC. LARGE-SCALE DATA N/A. LIMITATIONS, REASON FOR CAUTION As the reported frequency of EAOC is very low, we did not have access to those samples in our study. Moreover, by adopting a targeted gene array approach, we might have missed several other potentially-relevant genes associated with EAOC pathogenesis. The above panel of markers should be further validated in archived tissue samples from women with endometriosis who later in life developed EAOC. WIDER IMPLICATIONS OF THE FINDINGS Knowledge gained from this study, with further confirmation on EAOC cases, may help in developing screening methods to identify women with increased risk of EAOC. STUDY FUNDING/COMPETING INTEREST(S) The study is funded by the Swedish Research Council (2012-2844), a joint grant from Stockholm County and Karolinska Institutet (ALF), RGD network at Karolinska Institutet, Karolinska Institutet for doctoral education (KID), Estonian Ministry of Education and Research (IUT34-16), Enterprise Estonia (EU48695), Horizon 2020 innovation program (WIDENLIFE, 692065), European Union’s FP7 Marie Curie Industry-Academia Partnerships and Pathways funding (IAPP, SARM, EU324509) and MSCA-RISE-2015 project MOMENDO (691058). All authors have no competing interest.
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    C14orf132 gene is possibly related to extremely low birth weight
    (2016-09) Tiirats, Airi; Viltrop, Triin; Nõukas, Margit; Reimann, Ene; Salumets, Andres; Kõks, Sulev
    Background Despite extensive research the genetic component of extremely low birth weight (ELBW) in newborns has remained obscure. Results The aim of the case study was to identify candidate gene(s) causing ELBW in newborns and hypotrophy in infants. A family of four was studied: mother, father and two ELBW-phenotype children. Studies were made of the medical conditions of the second child at birth and post-partum - peculiar phenotype, micro-anomalies, recurrent infections, suspicion of autoimmune hepatitis, multifactorial encephalopathy and suspected metabolic and chromosomal abnormalities. Whole genome single nucleotide polymorphism (SNP) genotyping array was used to investigate the genomic rearrangements in both affected children using peripheral blood DNA samples. Whole blood transcriptome was assessed by using RNA sequencing (RNA-seq) in all four family members. RNA-seq identified a single gene – C14orf132 (chromosome 14 open reading frame 132) differentially expressed, with the level of the transcript significantly lower in the blood samples of the children. Copy number variant (CNV) analysis did not reveal any pathogenic CNVs in the region of C14orf132 gene of both affected children. Conclusion We demonstrated the importance of combining whole genome CNV and transcriptome analysis in identification of the candidate gene(s) in case studies. We propose the C14orf132 gene expression to be associated with the ELBW-phenotype. C14orf132 gene is a novel long non-coding RNA (lincRNA) with unknown function, which might be associated with the pre- and early postnatal developmental delay through the altered gene expression.
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    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.
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    Compartmentalized gene expression profiling of receptive endometrium reveals progesterone regulated ENPP3 is differentially expressed and secreted in glycosylated form
    (2016-09) Boggavarapu, Nageswara Rao; Lalitkumar, Sujata; Joshua, Vijay; Kasvandik, Sergo; Salumets, Andres; Lalit Kumar, Parameswaran Grace; Gemzell-Danielsson, Kristina
    The complexity of endometrial receptivity at the molecular level needs to be explored in detail to improve the management of infertility. Here, differential expression of transcriptomes in receptive endometrial glands and stroma revealed Ectonucleotide Pyrophosphatase/Phosphodiesterase 3 (ENPP3) as a progesterone regulated factor and confirmed by various methods, both at mRNA and protein level. The involvement of ENPP3 in embryo attachment was tested in an in vitro model for human embryo implantation. Interestingly, there was high expression of ENPP3 mRNA in stroma but not protein. Presence of N-glycosylated ENPP3 in receptive phase uterine fluid in women confirms its regulation by progesterone and makes it possible to use in a non-invasive test of endometrial receptivity.
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    Copy number variation analysis detects novel candidate genes involved in follicular growth and oocyte maturation in a cohort of premature ovarian failure cases
    (2016-06) Tšuiko, O.; Nõukas, M.; Žilina, O.; Hensen, K.; Tapanainen, J.; Mägi, R.; Kals, M.; Kivistik, PA.; Haller-Kikkatalo, K.; Salumets, A.; Kurg, A.
    STUDY QUESTION Can spontaneous premature ovarian failure (POF) patients derived from population-based biobanks reveal the association between copy number variations (CNVs) and POF? SUMMARY ANSWER CNVs can hamper the functional capacity of ovaries by disrupting key genes and pathways essential for proper ovarian function. WHAT IS KNOWN ALREADY POF is defined as the cessation of ovarian function before the age of 40 years. POF is a major reason for female infertility, although its cause remains largely unknown. STUDY DESIGN, SIZE, DURATION The current retrospective CNV study included 301 spontaneous POF patients and 3188 control individuals registered between 2003 and 2014 at Estonian Genome Center at the University of Tartu (EGCUT) biobank. PARTICIPANTS/MATERIALS, SETTING, METHODS DNA samples from 301 spontaneous POF patients were genotyped by Illumina HumanCoreExome (258 samples) and HumanOmniExpress (43 samples) BeadChip arrays. Genotype and phenotype information was drawn from the EGCUT for the 3188 control population samples, previously genotyped with HumanCNV370 and HumanOmniExpress BeadChip arrays. All identified CNVs were subjected to functional enrichment studies for highlighting the POF pathogenesis. Real-time quantitative PCR was used to validate a subset of CNVs. Whole-exome sequencing was performed on six patients carrying hemizygous deletions that encompass genes essential for meiosis or folliculogenesis. MAIN RESULTS AND THE ROLE OF CHANCE Eleven novel microdeletions and microduplications that encompass genes relevant to POF were identified. For example, FMN2 (1q43) and SGOL2 (2q33.1) are essential for meiotic progression, while TBP (6q27), SCARB1 (12q24.31), BNC1 (15q25) and ARFGAP3 (22q13.2) are involved in follicular growth and oocyte maturation. The importance of recently discovered hemizygous microdeletions of meiotic genes SYCE1 (10q26.3) and CPEB1 (15q25.2) in POF patients was also corroborated. LIMITATIONS, REASONS FOR CAUTION This is a descriptive analysis and no functional studies were performed. Anamnestic data obtained from population-based biobank lacked clinical, biological (hormone levels) or ultrasonographical data, and spontaneous POF was predicted retrospectively by excluding known extraovarian causes for premature menopause. WIDER IMPLICATIONS OF THE FINDINGS The present study, with high number of spontaneous POF cases, provides novel data on associations between the genomic aberrations and premature menopause of ovarian cause and demonstrates that population-based biobanks are powerful source of biological samples and clinical data to reveal novel genetic lesions associated with human reproductive health and disease, including POF. STUDY FUNDING/COMPETING INTEREST This study was supported by the Estonian Ministry of Education and Research (IUT20-43, IUT20-60, IUT34-16, SF0180027s10 and 9205), Enterprise Estonia (EU30020 and EU48695), Eureka's EUROSTARS programme (NOTED, EU41564), grants from European Union's FP7 Marie Curie Industry-Academia Partnerships and Pathways (IAPP, SARM, |EU324509) and Horizon 2020 innovation programme (WIDENLIFE, 692065), Academy of Finland and the Sigrid Juselius Foundation.
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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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    Demographic associations for autoantibodies in disease-free individuals of a European population
    (2016) Haller-Kikkatalo, Kadri; Alnek, Kristi; Metspalu, Andres; Mihailov, Evelin; Metsküla, Kaja; Kisand, Kalle; Pisarev, Heti; Salumets, Andres; Uibo, Raivo
    The presence of autoantibodies usually precedes autoimmune disease, but is sometimes considered an incidental finding with no clinical relevance. The prevalence of immune-mediated diseases was studied in a group of individuals from the Estonian Genome Project (n = 51,862), and 6 clinically significant autoantibodies were detected in a subgroup of 994 (auto)immune-mediated disease-free individuals. The overall prevalence of individuals with immune-mediated diseases in the primary cohort was 30.1%. Similarly, 23.6% of the participants in the disease-free subgroup were seropositive for at least one autoantibody. Several phenotypic parameters were associated with autoantibodies. The results suggest that (i) immune-mediated diseases are diagnosed in nearly one-third of a random European population, (ii) 6 common autoantibodies are detectable in almost one-third of individuals without diagnosed autoimmune diseases, (iii) tissue non-specific autoantibodies, especially at high levels, may reflect preclinical disease in symptom-free individuals, and (iv) the incidental positivity of anti-TPO in men with positive familial anamnesis of maternal autoimmune disease deserves further medical attention. These results encourage physicians to evaluate autoantibodies in addition to treating a variety of patient health complaints to detect autoimmune-mediated disease early.
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    Determination of biological activity of gonadotropins hCG and FSH by Förster resonance energy transfer based biosensors
    (2017) Mazina, Olga; Allikalt, Anni; Tapanainen, Juha S.; Salumets, Andres; Rinken, Ago
    Determination of biological activity of gonadotropin hormones is essential in reproductive medicine and pharmaceutical manufacturing of the hormonal preparations. The aim of the study was to adopt a G-protein coupled receptor (GPCR)-mediated signal transduction pathway based assay for quantification of biological activity of gonadotropins. We focussed on studying human chorionic gonadotropin (hCG) and follicle-stimulating hormone (FSH), as these hormones are widely used in clinical practice. Receptor-specific changes in cellular cyclic adenosine monophosphate (cAMP, second messenger in GPCR signalling) were monitored by a Förster resonance energy transfer (FRET) biosensor protein TEpacVV in living cells upon activation of the relevant gonadotropin receptor. The BacMam gene delivery system was used for biosensor protein expression in target cells. In the developed assay only biologically active hormones initiated GPCR-mediated cellular signalling. High assay sensitivities were achieved for detection of hCG (limit of detection, LOD: 5 pM) and FSH (LOD: 100 pM). Even the small-scale conformational changes caused by thermal inactivation and reducing the biological activity of the hormones were registered. In conclusion, the proposed assay is suitable for quantification of biological activity of gonadotropins and is a good alternative to antibody- and animal-testing-based assays used in pharmaceutical industry and clinical research.
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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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    DNA methylation alterations—potential cause of endometriosis pathogenesis or a reflection of tissue heterogeneity?
    (2018) Salumets, A.; Kaplinski, L.; Saare, M.; Krigul, KL.; Laisk-Podar, T.; Ponandai-Srinivasan, S.; Rahmioglu, N.; Lalit Kumar, PG.; Zondervan, K.; Peters, M.
    Alterations in the DNA methylation pattern of endometriotic lesions and endometrium of endometriosis patients have been proposed as one potential factor accompanying the endometriosis development. Although many differentially methylated genes have been associated with the pathogenesis of this disease, the overlap between the results of different studies has remained small. Among other potential confounders, the impact of tissue heterogeneity on the outcome of DNA methylation studies should be considered, as tissues are mixtures of different cell types with their own specific DNA methylation signatures. This review focuses on the results of DNA methylation studies in endometriosis from the cellular heterogeneity perspective. We consider both the studies using highly heterogeneous whole-lesion biopsies and endometrial tissue, as well as pure cell fractions isolated from lesions and endometrium to understand the potential impact of the cellular composition to the results of endometriosis DNA methylation studies. Also, future perspectives on how to diminish the impact of tissue heterogeneity in similar studies are provided.
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    DNA methylation changes in endometrium and correlation with gene expression during the transition from pre-receptive to receptive phase
    (2017) Kukushkina, V.; Modhukur, V.; Suhorutšenko, M.; Peters, M.; Mägi, R.; Rahmioglu, N.; Velthut-Meikas, A.; Altmäe, S.; Esteban, FJ.; Vilo, J.; Zondervan, K.; Salumets, A.; Laisk-Podar, T.
    The inner uterine lining (endometrium) is a unique tissue going through remarkable changes each menstrual cycle. Endometrium has its characteristic DNA methylation profile, although not much is known about the endometrial methylome changes throughout the menstrual cycle. The impact of methylome changes on gene expression and thereby on the function of the tissue, including establishing receptivity to implanting embryo, is also unclear. Therefore, this study used genome-wide technologies to characterize the methylome and the correlation between DNA methylation and gene expression in endometrial biopsies collected from 17 healthy fertile-aged women from pre-receptive and receptive phase within one menstrual cycle. Our study showed that the overall methylome remains relatively stable during this stage of the menstrual cycle, with small-scale changes affecting 5% of the studied CpG sites (22,272 out of studied 437,022 CpGs, FDR < 0.05). Of differentially methylated CpG sites with the largest absolute changes in methylation level, approximately 30% correlated with gene expression measured by RNA sequencing, with negative correlations being more common in 5′ UTR and positive correlations in the gene ‘Body’ region. According to our results, extracellular matrix organization and immune response are the pathways most affected by methylation changes during the transition from pre-receptive to receptive phase.
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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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    Genetic profiling could improve IVF success
    (Horizon, The EU Research&Innovation Magazine, 2018-03-29) Vermeeschm, Joris; Klotz, Frieda
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    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)
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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.