Competence Center on Reproductive Medicine and Biology

Tiigi, Estonia

Competence Center on Reproductive Medicine and Biology

Tiigi, Estonia
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Mandar R.,University of Tartu | Mandar R.,Competence Center on Reproductive Medicine and Biology
Pharmacological Research | Year: 2013

This manuscript describes the male genital tract microbiota and the significance of it on the host's and his partner's health. Microbiota exists in male lower genital tract, mostly in urethra and coronal sulcus while high inter-subject variability exists. Differences appear between sexually transmitted disease positive and negative men as well as circumcised and uncircumcised men. Upper genital tract is generally germ-free, except in case of infections. Prostatitis patients have frequently abundant polymicrobial communities in their semen, expressed prostatic secretion and/or post-massage urine. Coryneform bacteria have ambivalent role in male urogenital tract being frequently commensals but sometimes associated with prostatitis and urethritis. Interactions between male and female genital tract microbiota are highly likely yet there are very scarce studies on the couples' genital tract microbiota. Increase of bacterial vaginosis-type microbiota and coliforms are the most typical findings in men while the adverse effect of male genital tract bacteria on in vitro fertilization and pregnancy outcome has also been indicated. © 2012 Elsevier Ltd. All Rights Reserved.

Velthut-Meikas A.,Competence Center on Reproductive Medicine and Biology | Velthut-Meikas A.,Tallinn University of Technology | Velthut-Meikas A.,University of Tartu | Simm J.,Tallinn University of Technology | And 7 more authors.
Molecular Endocrinology | Year: 2013

The granulosa cells in the mammalian ovarian follicle respond to gonadotropin signaling and are involved in the processes of folliculogenesis and oocyte maturation. Studies on gene expression and regulation in human granulosa cells are of interest due to their potential for estimating the oocyte viability and in vitro fertilization success. However, the posttranscriptional gene expression studies on micro-RNA (miRNA) level in the human ovary have been scarce. The current study determined the miRNA profile by deep sequencing of the 2 intrafollicular somatic cell types: mural and cumulus granulosa cells (MGCs and CGCs, respectively) isolated from women undergoing controlled ovarian stimulation and in vitro fertilization. Altogether, 936 annotated and 9 novel miRNAs were identified. Ninety of the annotated miRNAs were differentially expressed between MGCs and CGCs. Bioinformatic prediction revealed that TGFβ, ErbB signaling, and heparan sulfate biosynthesis were targeted by miRNAs in both granulosa cell populations, whereas extracellular matrix remodeling, Wnt, and neurotrophin signaling pathways were enriched among miRNA targets in MGCs. Two of the nine novel miRNAs found were of intronic origin: one from the aromatase and the other from the FSH receptor gene. The latter miRNA was predicted to target the activin signaling pathway. In addition to revealing the genome-wide miRNA signature in human granulosa cells, our results suggest that posttranscriptional regulation of gene expression by miRNAs could play an important role in the modification of gonadotropin signaling. miRNA expression studies could therefore lead to new prognostic markers in assisted reproductive technologies. © 2013 by The Endocrine Society.

Altmae S.,Competence Center on Reproductive Medicine and Biology | Altmae S.,University of Granada | Martinez-Conejero J.A.,IVIOMICS | Esteban F.J.,University of Jaén | And 5 more authors.
Reproductive Sciences | Year: 2013

MicroRNAs (miRNAs) act as important epigenetic posttranscriptional regulators of gene expression. We aimed to gain more understanding of the complex gene expression regulation of endometrial receptivity by analyzing miRNA signatures of fertile human endometria. We set up to analyze miRNA signatures of receptive (LH + 7, n = 4) versus prereceptive (LH + 2, n = 5) endometrium from healthy fertile women. We found hsa-miR-30b and hsa-miR-30d to be significantly upregulated, and hsa-miR-494 and hsa-miR-923 to be downregulated in receptive endometrium. Three algorithms (miRanda, PicTar, and TargetScan) were used for target gene prediction. Functional analyses of the targets using Ingenuity Pathways Analysis and The Database for Annotation, Visualization and Integrated Discovery indicated roles in transcription, cell proliferation and apoptosis, and significant involvement in several relevant pathways, such as axon guidance, Wnt/β-catenin, ERK/MAPK, transforming growth factor β (TGF-β), p53 and leukocyte extravasation. Comparison of predicted miRNA target genes and our previous messenger RNA microarray data resulted in a list of 12 genes, including CAST, CFTR, FGFR2, and LIF that could serve as a panel of genes important for endometrial receptivity. In conclusion, we suggest that a subset of miRNAs and their target genes may play important roles in endometrial receptivity. © 2013 The Author(s).

Tontson L.,University of Tartu | Kopanchuk S.,University of Tartu | Kopanchuk S.,Competence Center on Reproductive Medicine and Biology | Rinken A.,University of Tartu | Rinken A.,Competence Center on Reproductive Medicine and Biology
Neurochemistry International | Year: 2014

Bodipy-FL-NAN-190 was found to be well suited for characterization of ligand binding to 5-HT1A receptors expressed in budded baculovirus particles, as binding is accompanied by large increases in fluorescence intensity and anisotropy. This ligand appears to bind rapidly (t 1/2,ass < 1 min), reversibly (t1/2,diss ∼ 6 min) and has high affinity (Kd = 0.30 ± 0.13 nM). This fluorescence anisotropy assay based on Bodipy-FL-NAN-190 binding to baculovirus particles was also a suitable assay system for the pharmacological characterization of non-labelled serotonergic ligands, as well as being sensitive to the presence of G-proteins and guanine nucleotides. Coexpression of αi subunits of human G-proteins in baculovirus particles resulted in the appearance of significantly greater proportion of nucleotide sensitive high affinity agonist binding sites. There were no significant differences between αi1 and αi3 subtypes, while ligand binding in the presence of αi2 had higher sensitivity to GDP and Mn2+.© 2014 Elsevier B.V.

Altmae S.,Karolinska University Hospital | Altmae S.,Competence Center on Reproductive Medicine and Biology | Hovatta O.,Karolinska University Hospital | Stavreus-Evers A.,Uppsala University | And 2 more authors.
Human Reproduction Update | Year: 2011

Background: Nowadays, the use of IVF has improved the prospects of infertility treatment. The expected outcome of IVF depends greatly on the effectiveness of controlled ovarian hyperstimulation (COH), where exogenous gonadotrophins are used to induce folliculogenesis. The response to stimulation varies substantially among women and is difficult to predict. Several predictive markers of COH outcome have been proposed (e.g. maternal age and ovarian reserve), but the search for optimal predictors is ongoing. Pharmacogenetic studies demonstrate the effects of individual genetic variability on COH outcome and the potential for customizing therapy based on the patient's genome. Methods: MEDLINE, EMBASE, DARE, CINAHL and the Cochrane Library, and references from relevant articles were investigated up to February 2011 regarding any common genetic variation and COH/IVF outcome. Results: Several polymorphisms in genes involved in FSH signalling, estrogen biosynthesis, folliculogenesis, folatemetabolismand other aspects influence the response to exogenous gonadotrophin administration, resulting in differences in COH and IVF outcomes. Nevertheless, the most studied polymorphism FSHR Asn680Ser is practically the only genetic marker, together with ESR1 PvuII T/C, that could be applied in clinical tests. Conclusions: Although data are accumulating with evidence suggesting that the ovarian response to COH is mediated by various polymorphisms, the optimal biomarkers and the efficacy of the tests still remain to be evaluated. © The Author 2011. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.

Haller-Kikkatalo K.,University of Tartu | Haller-Kikkatalo K.,Competence Center on Reproductive Medicine and Biology | Salumets A.,University of Tartu | Salumets A.,Competence Center on Reproductive Medicine and Biology | And 2 more authors.
Clinical and Developmental Immunology | Year: 2012

Female fertility can be affected by diseases or dysfunctions of reproductive tract, neuroendocrine system, and immune system. Reproductive autoimmune failure can be associated with overall activation of immune system or with immune system reactions specifically directed against ovarian antigens. Majority of the antiovarian autoantibodies are directed against -subunit of follicle stimulating hormone (anti-FSH). This paper summarizes a current clinical classification of female infertility in the context of general activation of autoimmunity and antiovarian autoimmunity by describing serum anti-FSH. The presence of naturally occurring anti-FSH in healthy women will be discussed. In addition, the putative impairment of ovarian folliculogenesis in case of increased production of those antibodies in infertile women will be characterized. Copyright © 2012 Kadri Haller-Kikkatalo et al.

Veiksina S.,University of Tartu | Veiksina S.,Competence Center on Reproductive Medicine and Biology | Kopanchuk S.,University of Tartu | Kopanchuk S.,Competence Center on Reproductive Medicine and Biology | And 2 more authors.
Biochimica et Biophysica Acta - Biomembranes | Year: 2014

We present here the implementation of budded baculoviruses that display G protein-coupled receptors on their surfaces for the investigation of ligand-receptor interactions using fluorescence anisotropy (FA). Melanocortin 4 (MC4) receptors and the fluorescent ligand Cy3B-NDP-α-MSH were used as the model system. The real-time monitoring of reactions and the high assay quality allow the application of global data analysis with kinetic mechanistic models that take into account the effect of nonspecific interactions and the depletion of the fluorescent ligand during the reaction. The receptor concentration, affinity and kinetic parameters of fluorescent ligand binding as well as state anisotropies for different fluorescent ligand populations were determined. At low Cy3B-NDP-α-MSH concentrations, a one-site receptor-ligand binding model described the processes, whereas divergence from this model was observed at higher ligand concentrations, which indicated a more complex mechanism of interactions similar to those mechanisms that have been found in experiments with radioactive ligands. The information obtained from our kinetic experiments and the inherent flexibility of FA assays also allowed the estimation of binding parameters for several MC4 receptor-specific unlabelled compounds. In summary, the FA assay that was developed with budded baculoviruses led the experimental data to a level that would solve complex models of receptor-ligand interactions also for other receptor systems and would become as a valuable tool for the screening of pharmacologically active compounds. © 2013 Elsevier B.V.

Nolvak H.,University of Tartu | Nolvak H.,Competence Center on Reproductive Medicine and Biology | Truu M.,University of Tartu | Truu J.,Competence Center on Reproductive Medicine and Biology | Truu J.,University of Tartu
Science of the Total Environment | Year: 2012

The study examined the variability in 16S ribosomal RNA (16S rRNA) and tetracycline resistance tetA gene quantification from environmental samples in relation to modifications in quantitative polymerase chain reaction (qPCR) workflow and subsequent data evaluation and analysis. We analysed three types of soil samples using two DNA extraction methods, two qPCR chemistries (SYBR green, LUX ™), and qPCR reaction kits from different manufacturers. To improve data quality, we employed a three-step amplification outlier removal approach prior to gene quantification calculations. We compared three variants of target gene enumerations and four variants of functional tetA gene normalisations against 16S rRNA genes. Results reveal that modifications in qPCR workflow steps significantly influence the gene quantification results from environmental samples. Primary factors affecting qPCR amplification efficiency included the variability of the target amplicon and the qPCR chemistry; the quality of the resulting datasets also had an impact. Although LUX ™ qPCR has shown promise for environmental samples, SYBR green qPCR yielded considerably better-quality datasets and higher, more stable amplification efficiency values. Gene enumeration data of outlier-removed and unmodified sample sets showed minor differences for good-quality datasets (i.e., amplifications with SYBR green), but differed by up to 40% among lower-quality datasets. Different DNA extraction methods yielded varying amounts and purities of extracted microbial community DNA from environmental samples, with as much as an order of magnitude variation in gene copy numbers. Target gene normalisations yielded stable results on good-quality data, regardless of the DNA extraction method or qPCR chemistry used. Even though qPCR is regarded as a precise method with low detection limit, technical variability in the qPCR workflow tends to overestimate or effectively mask minute changes in community. © 2012 Elsevier B.V.

Lokk K.,University of Tartu | Modhukur V.,University of Tartu | Rajashekar B.,University of Tartu | Martens K.,University of Tartu | And 8 more authors.
Genome Biology | Year: 2014

Background: DNA epigenetic modifications, such as methylation, are important regulators of tissue differentiation, contributing to processes of both development and cancer. Profiling the tissue-specific DNA methylome patterns will provide novel insights into normal and pathogenic mechanisms, as well as help in future epigenetic therapies. In this study, 17 somatic tissues from four autopsied humans were subjected to functional genome analysis using the Illumina Infinium HumanMethylation450 BeadChip, covering 486 428 CpG sites. Results: Only 2% of the CpGs analyzed are hypermethylated in all 17 tissue specimens; these permanently methylated CpG sites are located predominantly in gene-body regions. In contrast, 15% of the CpGs are hypomethylated in all specimens and are primarily located in regions proximal to transcription start sites. A vast number of tissue-specific differentially methylated regions are identified and considered likely mediators of tissue-specific gene regulatory mechanisms since the hypomethylated regions are closely related to known functions of the corresponding tissue. Finally, a clear inverse correlation is observed between promoter methylation within CpG islands and gene expression data obtained from publicly available databases. Conclusions: This genome-wide methylation profiling study identified tissue-specific differentially methylated regions in 17 human somatic tissues. Many of the genes corresponding to these differentially methylated regions contribute to tissue-specific functions. Future studies may use these data as a reference to identify markers of perturbed differentiation and disease-related pathogenic mechanisms. © 2014 Lokk et al.; licensee BioMed Central Ltd.

Altmae S.,Karolinska University Hospital | Altmae S.,Competence Center on Reproductive Medicine and Biology | Salumets A.,Competence Center on Reproductive Medicine and Biology
Reproductive BioMedicine Online | Year: 2011

Male factor infertility is a growing problem worldwide. Considering that a male factor is involved in at least 20% of infertility cases, there is a need for better predictive markers of sperm function. The traditional sperm analysis based on sperm count and motility has been used for the diagnosis of male fertility for several decades, however, a significant number of men with normal sperm features remain unable to reach pregnancy. This fact clearly indicates the need to develop new male infertility tests to accurately diagnose the sperm samples from these individuals. Furthermore, the classic spermiogram has limited predictive power to predict pregnancy in assisted reproductive techniques. Microarray technology is a powerful tool for detecting gene expression of thousands of genes at the same time. There is a great interest in the sperm transcriptome as a source from which to develop markers of male infertility. This commentary discusses the current advances in the microarray technology and sperm quality. It is believed that microarray-based fertility testing of sperm potential in infertility treatment could be close at hand. © 2011, Reproductive Healthcare Ltd. All rights reserved.

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