Merli Saare

Comparison of serum exosome isolation methods for microRNA profiling.

OBJECTIVES Exosomes are small membrane bound vesicles secreted by most cell types. Exosomes contain various functional proteins, mRNAs and microRNAs (miRNAs) that could be used for diagnostic and therapeutic purposes. Currently, a standard method for serum exosome isolation is differential ultracentrifugation, but a search for alternative, less time-consuming and labour extensive exosomal isolation method for use in clinical settings is ongoing. The effect of serum exosome isolation method on obtained miRNA profile is not yet clear. The aim of this study was to determine to which extent selected exosome isolation methods influence the serum exosomal miRNA profile. DESIGN AND METHODS Exosomes were isolated from blood serum of healthy individuals by ultracentrifugation and ExoQuick Precipitation methods. The expression profile of 375 miRNAs was determined by real time PCR using Exiqon miRCURY LNA™ microRNA Human panel I assays. RESULTS Although a strong correlation of exosomal miRNA profiles was observed between the two isolation methods, distinct clusters of miRNA levels between the used methods were identified. The detected levels of two miRNAs, miR-92a and miR-486-5p, were significantly influenced by the exosome isolation method used. CONCLUSIONS Both exosome isolation methods are suitable for serum exosomal miRNA profiling. Differences found in miRNA patterns between the two methods indicate that the observed exosomal miRNA profile is slightly affected by the extracellular vesicle isolation method.

Research Resource: Interactome of Human Embryo Implantation: Identification of Gene Expression Pathways, Regulation, and Integrated Regulatory Networks

A prerequisite for successful embryo implantation is adequate preparation of receptive endometrium and the establishment and maintenance of a viable embryo. The success of implantation further relies upon a two-way dialogue between the embryo and uterus. However, molecular bases of these preimplantation and implantation processes in humans are not well known. We performed genome expression analyses of human embryos (n = 128) and human endometria (n = 8). We integrated these data with protein-protein interactions in order to identify molecular networks within the endometrium and the embryo, and potential embryo-endometrium interactions at the time of implantation. For that, we applied a novel network profiling algorithm HyperModules, which combines topological module identification and functional enrichment analysis. We found a major wave of transcriptional down-regulation in preimplantation embryos. In receptive-stage endometrium, several genes and signaling pathways were identified, including JAK-STAT signaling and inflammatory pathways. The main curated embryo-endometrium interaction network highlighted the importance of cell adhesion molecules in the implantation process. We also identified cytokine-cytokine receptor interactions involved in implantation, where osteopontin (SPP1), leukemia inhibitory factor (LIF) and leptin (LEP) pathways were intertwining. Further, we identified a number of novel players in human embryo-endometrium interactions, such as apolipoprotein D (APOD), endothelin 1 (END1), fibroblast growth factor 7 (FGF7), gastrin (GAST), kringle containing trnasmembrane protein 1 (KREMEN1), neuropilin 1 (NRP1), serpin peptidase inhibitor clade A member 3 (SERPINA3), versican (VCAN), and others. Our findings provide a fundamental resource for better understanding of the genetic network that leads to successful embryo implantation. We demonstrate the first systems biology approach into the complex molecular network of the implantation process in humans.

Polymorphisms in MMP-2 and MMP-9 promoter regions are associated with endometriosis

In this case-control study, we investigated the potential associations of MMP-2 and MMP-9 gene promoter region polymorphisms as well as MMP-2 promoter haplotypes with susceptibility to endometriosis in women of caucasian origin. The results demonstrated that polymorphisms in MMP-2 (-735 C/T) and MMP-9 (-1562 C/T) were associated with elevated risk of endometriosis and that certain MMP-2 promoter haplotypes were more common in control group.

Circulating microRNA Profile throughout the menstrual cycle.

Normal physiological variables, such as age and gender, contribute to alterations in circulating microRNA (miRNA) expression levels. The changes in the female body during the menstrual cycle can also be reflected in plasma miRNA expression levels. Therefore, this study aimed to determine the plasma miRNA profile of healthy women during the menstrual cycle and to assess which circulating miRNAs are derived from blood cells. The plasma miRNA expression profiles in nine healthy women were determined by quantitative real time PCR using Exiqon Human Panel I assays from four time-points of the menstrual cycle. This platform was also used for studying miRNAs from pooled whole blood RNA samples at the same four time-points. Our results indicated that circulating miRNA expression levels in healthy women were not significantly altered by the processes occurring during the menstrual cycle. No significant differences in plasma miRNA expression levels were observed between the menstrual cycle time-points, but the number of detected miRNAs showed considerable variation among the studied individuals. miRNA analysis from whole blood samples revealed that majority of miRNAs in plasma are derived from blood cells. The most abundant miRNA in plasma and blood was hsa-miR-451a, but a number of miRNAs were only detected in one or the other sample type. In conclusion, our data suggest that the changes in the female body during the menstrual cycle do not affect the expression of circulating miRNAs at measurable levels.

Circulating miR-200–family micro-RNAs have altered plasma levels in patients with endometriosis and vary with blood collection time

OBJECTIVE To determine whether circulating micro-RNA (miR) 200a, miR-200b, and miR-141 have altered levels in patients with endometriosis compared with control individuals. DESIGN Experimental laboratory study. SETTING University. PATIENT(S) Patients with endometriosis (n = 61), laparoscopically confirmed endometriosis-free women (n = 35), and self-reported healthy women (n = 30) were included in the study. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Plasma miRNA levels in endometriosis patients and control subjects. RESULT(S) We found that the levels of studied miRNAs varied with blood collection time, being lower in the morning than in the evening. When blood collection time was taken into account, the results revealed significantly lower levels of miR-200a and miR-141 in the evening plasma samples of women with endometriosis compared with surgically confirmed disease-free patients. However, the evening-sample levels of all three miRNAs were significantly lower in patients with stage I-II endometriosis than in endometriosis-free control subjects. In cases of stage III-IV endometriosis, only miR-200a levels were significantly lower compared with patients without endometriosis. Circulating miR-200a showed the best discriminative power to differentiate women with endometriosis from patients with similar complaints but without the disease. CONCLUSION(S) Our findings suggest that miR-200a and miR-141 have a potential as novel noninvasive biomarkers for endometriosis. In addition, we found that the plasma miR-200a, miR-200b and miR-141 levels vary with blood sampling time, so it is important to take the sample collection time into account when studying miRNAs as biomarkers.

Aromatase gene (CYP19A1) variants, female infertility and ovarian stimulation outcome: a preliminary report

Progress has been made towards ascertaining the genetic predictors of ovarian stimulation in IVF. Aromatase cytochrome P450, encoded by the CYP19A1 gene, catalyses a key step in ovarian oestrogen biosynthesis. Hence, the aromatase gene is an attractive candidate for genetic studies. This study aimed to examine the genetic influences of CYP19A1 TCT trinucleotide insertion/deletion (Ins/Del) and (TTTA)(n) microsatellite intronic polymorphisms on ovarian stimulation outcome and aetiology of female infertility. IVF patients (n = 152) underwent ovarian stimulation according to recombinant FSH and gonadotrophin releasing hormone antagonist protocol. Del/Del homozygous patients with shorter TTTA repeats exhibited decreased ovarian FSH sensitivity in ovarian stimulation, which may reflect variations in aromatase gene expression during early antral follicle development. Accordingly, this study demonstrates correlations between Del allele and shorter (TTTA)(n) repeat sizes with smaller ovaries (r = -0.70, P = 0.047) and fewer antral follicles (r = 0.21, P = 0.018) on days 3-5 of spontaneous menstrual cycle, respectively. Furthermore, Del variation linked with low-repeat-number (TTTA)(n) alleles are involved in enhanced genetic susceptibility to unexplained infertility (adjusted OR = 4.33, P = 0.039) and endometriosis (r = -0.88, P = 0.026), which corroborates evidence on the overlapping patient profiles of ovarian dysfunction in both types of female infertility.

Association of CCR5, TLR2, TLR4 and MBL genetic variations with genital tract infections and tubal factor infertility.

Upper genital tract infections can inflict inadequate immune response and cause Fallopian tube damage and concomitant female infertility. However, the exact role of host genetic variation in the development of tubal factor infertility remains unclear. We selected nine genetic variations in four genes involved in immune response modulation (CCR5, TLR2, TLR4 and MBL2) and assessed their association with tubal factor infertility by comparing genotype frequencies among 163 women with tubal factor infertility and 400 control individuals. The CCR5, TLR2 and TLR4 genotypes were not associated with tubal factor infertility, although the TLR4 Asp299Gly and Thr399Ile heterozygosity was associated with a decreased incidence of pathogens associated with genital tract infections in tubal factor infertility patients. In contrast, MBL2 low-producing genotypes were associated with an increased incidence of such pathogens. In addition, hyper-producing MBL2 genotype HYA/HYA and low-producing MBL2 genotypes were associated with susceptibility to tubal factor infertility, while a protective effect was associated with the high-producing MBL2 genotype HYA/LYA. Overall, these data suggest that polymorphisms in TLR4 and MBL2 play a role in receptiveness to pathogens causing genital tract infections, while MBL2 genotypes contribute to susceptibility to tubal factor infertility.

High-Throughput Sequencing Approach Uncovers the miRNome of Peritoneal Endometriotic Lesions and Adjacent Healthy Tissues

Accumulating data have shown the involvement of microRNAs (miRNAs) in endometriosis pathogenesis. In this study, we used a novel approach to determine the endometriotic lesion-specific miRNAs by high-throughput small RNA sequencing of paired samples of peritoneal endometriotic lesions and matched healthy surrounding tissues together with eutopic endometria of the same patients. We found five miRNAs specific to epithelial cells – miR-34c, miR-449a, miR-200a, miR-200b and miR-141 showing significantly higher expression in peritoneal endometriotic lesions compared to healthy peritoneal tissues. We also determined the expression levels of miR-200 family target genes E-cadherin, ZEB1 and ZEB2 and found that the expression level of E-cadherin was significantly higher in endometriotic lesions compared to healthy tissues. Further evaluation verified that studied miRNAs could be used as diagnostic markers for confirming the presence of endometrial cells in endometriotic lesion biopsy samples. Furthermore, we demonstrated that the miRNA profile of peritoneal endometriotic lesion biopsies is largely masked by the surrounding peritoneal tissue, challenging the discovery of an accurate lesion-specific miRNA profile. Taken together, our findings indicate that only particular miRNAs with a significantly higher expression in endometriotic cells can be detected from lesion biopsies, and can serve as diagnostic markers for endometriosis.

Genetic variations in vascular endothelial growth factor but not in angiotensin I-converting enzyme genes are associated with endometriosis in Estonian women

OBJECTIVE To determine plausible associations between endometriosis and vascular endothelial growth factor gene (VEGF -2578 A/C, -1154 G/A, -634 G/C and 936 C/T), also angiotensin I-converting enzyme gene (ACE -240 A/T and 2350 A/G) single nucleotide polymorphisms (SNPs), as well as their respective haplotypes. STUDY DESIGN PCR-based restriction fragment length polymorphism analysis was used to detect SNPs in VEGF and ACE genes in 150 Estonian women with endometriosis and 199 control subjects. RESULTS The CC genotype of the VEGF -2578 A/C SNP was correlated with a decreased risk of endometriosis (OR=0.40, 95% CI 0.20-0.78). Other VEGF and ACE SNPs and haplotypes were not associated with endometriosis. CONCLUSION This case-control study demonstrated that the VEGF -2578 A/C SNP may influence susceptibility to endometriosis in the Estonian population, while associations between endometriosis and other VEGF and ACE SNPs, as well as the respective haplotypes are unlikely.

The influence of menstrual cycle and endometriosis on endometrial methylome.

BackgroundAlterations in endometrial DNA methylation profile have been proposed as one potential mechanism initiating the development of endometriosis. However, the normal endometrial methylome is influenced by the cyclic hormonal changes, and the menstrual cycle phase-dependent epigenetic signature should be considered when studying endometrial disorders. So far, no studies have been performed to evaluate the menstrual cycle influences and endometriosis-specific endometrial methylation pattern at the same time.ResultsInfinium HumanMethylation 450K BeadChip arrays were used to explore DNA methylation profiles of endometrial tissues from various menstrual cycle phases from 31 patients with endometriosis and 24 healthy women. The DNA methylation profile of patients and controls was highly similar and only 28 differentially methylated regions (DMRs) between patients and controls were found. However, the overall magnitude of the methylation differences between patients and controls was rather small (Δβ ranging from –0.01 to –0.16 and from 0.01 to 0.08, respectively, for hypo- and hypermethylated CpGs). Unsupervised hierarchical clustering of the methylation data divided endometrial samples based on the menstrual cycle phase rather than diseased/non-diseased status. Further analysis revealed a number of menstrual cycle phase-specific epigenetic changes with largest changes occurring during the late-secretory and menstrual phases when substantial rearrangements of endometrial tissue take place. Comparison of cycle phase- and endometriosis-specific methylation profile changes revealed that 13 out of 28 endometriosis-specific DMRs were present in both datasets.ConclusionsThe results of our study accentuate the importance of considering normal cyclic epigenetic changes in studies investigating endometrium-related disease-specific methylation patterns.