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.
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.
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.
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.
•miRNA profiles obtained by two exosomal isolation methods were compared.•Used exosome isolation methods are suitable for serum exosomal miRNA profiling.•Exosomal miRNA profile is somewhat dependent upon the exosome isolation method.•Higher miRNA yield in serum exosomes was detected by ExoQuick method.
Previous transcriptome studies of the human endometrium have revealed hundreds of simultaneously up- and down-regulated genes that are involved in endometrial receptivity. However, the overlap ...between the studies is relatively small, and we are still searching for potential diagnostic biomarkers. Here we perform a meta-analysis of endometrial-receptivity associated genes on 164 endometrial samples (76 from 'pre-receptive' and 88 from mid-secretory, 'receptive' phase endometria) using a robust rank aggregation (RRA) method, followed by enrichment analysis, and regulatory microRNA prediction. We identify a meta-signature of endometrial receptivity involving 57 mRNA genes as putative receptivity markers, where 39 of these we confirm experimentally using RNA-sequencing method in two separate datasets. The meta-signature genes highlight the importance of immune responses, the complement cascade pathway and the involvement of exosomes in mid-secretory endometrial functions. Bioinformatic prediction identifies 348 microRNAs that could regulate 30 endometrial-receptivity associated genes, and we confirm experimentally the decreased expression of 19 microRNAs with 11 corresponding up-regulated meta-signature genes in our validation experiments. The 57 identified meta-signature genes and involved pathways, together with their regulatory microRNAs could serve as promising and sought-after biomarkers of endometrial receptivity, fertility and infertility.
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.
Primary ovarian insufficiency (POI) affects ∼1% of women before 40 years of age. The recent leap in genetic knowledge obtained by next generation sequencing (NGS) together with animal models has ...further elucidated its molecular pathogenesis, identifying novel genes/pathways. Mutations of >60 genes emphasize high genetic heterogeneity. Genome-wide association studies have revealed a shared genetic background between POI and reproductive aging. NGS will provide a genetic diagnosis leading to genetic/therapeutic counseling: first, defects in meiosis or DNA repair genes may predispose to tumors; and second, specific gene defects may predict the risk of rapid loss of a persistent ovarian reserve, an important determinant in fertility preservation. Indeed, a recent innovative treatment of POI by in vitro activation of dormant follicles proved to be successful.
The mechanisms underlying the formation of the ovarian reserve are generally well conserved, from Drosophila to mammals. Owing to this high degree of conservation, factors shown to regulate the ovarian reserve in mouse models are all potential candidates for identifying mutations associated with POI in humans.
With the generation of genetically modified mice, much insight has been gained into the mechanisms that control the formation of the ovarian reserve and trigger the activation of primordial follicles.
Comparison with animal models is complicated by the fact that the phenotype of complete gene deletion in knockout models may not be mimicked by single gene mutations.
Recently innovative treatment for POI based on in vitro activation of the dormant primordial follicular pool has been developed.
In mammalian species an optimal fertilization window during which successful fertilization occurs. In the majority of mammals estrus marks ovulation time and coincident with mating, thereby allowing ...the synchronized meeting in the fallopian tubes, between freshly ejaculated sperm and freshly ovulated oocytes. Conversely, women do not show natural visual signs of ovulation such that fertilization can occur hours later involving an aged oocyte and freshly ejaculated spermatozoa. During this time, the oocyte undergoes a rapid degradation known as “postovulatory aging” (POA). POA may become particularly important in the human-assisted reproductive technologies, as the fertilization of retrieved mature oocytes can be delayed due to increased laboratory workload or because of unforeseeable circumstances, like the delayed availability of semen samples. This paper is an updated review of the consequences of POA, either
in vivo
or
in vitro
, on oocyte quality with particular attention to modifications caused by POA on oocyte nuclear, cytoplasmic, genomic, and epigenetic maturation, and embryo development.
The application of high-throughput sequencing methods has raised doubt in the concept of the uniform healthy vaginal microbiota consisting predominantly of lactobacilli by revealing the existence of ...more variable bacterial community composition. As this needs to be analyzed more extensively and there is little straightforward data regarding the vaginal mycobiome of asymptomatic women we aimed to define bacterial and fungal communities in vaginal samples from 494 asymptomatic, reproductive-age Estonian women. The composition of the vaginal microbiota was determined by amplifying bacterial 16S rRNA and fungal internal transcribed spacer-1 (ITS-1) regions and subsequently sequencing them using 454 Life Sciences pyrosequencing. We delineated five major bacterial community groups with distinctive diversity and species composition. Lactobacilli were among the most abundant bacteria in all groups, but also members of genus Gardnerella had high relative abundance in some of the groups. Microbial diversity increased with higher vaginal pH values, and was also higher when a malodorous discharge was present, indicating that some of the women who consider themselves healthy may potentially have asymptomatic bacterial vaginosis (BV). Our study is the first of its kind to analyze the mycobiome that colonizes the healthy vaginal environment using barcoded pyrosequencing technology. We observed 196 fungal operational taxonomic units (OTUs), including 16 OTUs of Candida spp., which is more diverse than previously recognized. However, assessing true fungal diversity was complicated because of the problems regarding the possible air-borne contamination and bioinformatics used for identification of fungal taxons as significant proportion of fungal sequences were assigned to unspecified OTUs.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Hyperandrogenic women with PCOS show disrupted decidualization (DE) and placentation. Dihydrotestosterone (DHT) is reported to enhance DE in non-PCOS endometrial stromal cells (eSC
Ctrl
); ...however, this has not been assessed in PCOS cells (eSC
PCOS
). Therefore, we studied the transcriptome profile of non-decidualized (non-DE) and DE eSCs from women with PCOS and Ctrl in response to short-term estradiol (E2) and/or progesterone (P4) exposure with/without (±) DHT. The non-DE eSCs were subjected to E2 ± DHT treatment, whereas the DE (0.5 mM 8-Br-cAMP, 96 h) eSCs were post-treated with E2 and P4 ± DHT, and RNA-sequenced. Validation was performed by immunofluorescence and immunohistochemistry. The results showed that, regardless of treatment, the PCOS and Ctrl samples clustered separately. The comparison of DE vs
.
non-DE eSC
PCOS
without DHT revealed PCOS-specific differentially expressed genes (DEGs) involved in mitochondrial function and progesterone signaling. When further adding DHT, we detected altered responses for lysophosphatidic acid (LPA), inflammation, and androgen signaling
.
Overall, the results highlight an underlying defect in decidualized eSC
PCOS
, present with or without DHT exposure, and possibly linked to the altered pregnancy outcomes. We also report novel factors which elucidate the mechanisms of endometrial dysfunction in PCOS.
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.
Endometriosis is a debilitating and difficult-to-diagnose gynecological disease. Owing to limited information and awareness, women often rely on social media platforms as a support system to engage ...in discussions regarding their disease-related concerns.
This study aimed to apply computational techniques to social media posts to identify discussion topics about endometriosis and to identify themes that require more attention from health care professionals and researchers. We also aimed to explore whether, amid the challenging nature of the disease, there are themes within the endometriosis community that gather posts with positive sentiments.
We retrospectively extracted posts from the subreddits r/Endo and r/endometriosis from January 2011 to April 2022. We analyzed 45,693 Reddit posts using sentiment analysis and topic modeling-based methods in machine learning.
Since 2011, the number of posts and comments has increased steadily. The posts were categorized into 11 categories, and the highest number of posts were related to either asking for information (Question); sharing the experiences (Rant/Vent); or diagnosing and treating endometriosis, especially surgery (Surgery related). Sentiment analysis revealed that 92.09% (42,077/45,693) of posts were associated with negative sentiments, only 2.3% (1053/45,693) expressed positive feelings, and there were no categories with more positive than negative posts. Topic modeling revealed 27 major topics, and the most popular topics were Surgery, Questions/Advice, Diagnosis, and Pain. The Survey/Research topic, which brought together most research-related posts, was the last in terms of posts.
Our study shows that posts on social media platforms can provide insights into the concerns of women with endometriosis symptoms. The analysis of the posts confirmed that women with endometriosis have to face negative emotions and pain daily. The large number of posts related to asking questions shows that women do not receive sufficient information from physicians and need community support to cope with the disease. Health care professionals should pay more attention to the symptoms and diagnosis of endometriosis, discuss these topics with patients to reduce their dissatisfaction with doctors, and contribute more to the overall well-being of women with endometriosis. Researchers should also become more involved in social media and share new science-based knowledge regarding endometriosis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Osteogenesis Imperfecta (OI) is a rare genetic disorder involving bone fragility. OI patients typically suffer from numerous fractures, skeletal deformities, shortness of stature and hearing loss. ...The disorder is characterised by genetic and clinical heterogeneity. Pathogenic variants in more than 20 different genes can lead to OI, and phenotypes can range from mild to lethal forms. As a genetic disorder which undoubtedly affects quality of life, OI significantly alters the reproductive confidence of families at risk. The current review describes a selection of the latest reproductive approaches which may be suitable for prospective parents faced with a risk of OI. The aim of the review is to alleviate suffering in relation to family planning around OI, by enabling prospective parents to make informed and independent decisions.
The current review provides a comprehensive overview of possible reproductive options for people with OI and for unaffected carriers of OI pathogenic genetic variants. The review considers reproductive options across all phases of family planning, including pre-pregnancy, fertilisation, pregnancy, and post-pregnancy. Special attention is given to the more modern techniques of assisted reproduction, such as preconception carrier screening, preimplantation genetic testing for monogenic diseases and non-invasive prenatal testing. The review outlines the methodologies of the different reproductive approaches available to OI families and highlights their advantages and disadvantages. These are presented as a decision tree, which takes into account the autosomal dominant and autosomal recessive nature of the OI variants, and the OI-related risks of people without OI. The complex process of decision-making around OI reproductive options is also discussed from an ethical perspective.
The rapid development of molecular techniques has led to the availability of a wide variety of reproductive options for prospective parents faced with a risk of OI. However, such options may raise ethical concerns in terms of methodologies, choice management and good clinical practice in reproductive care, which are yet to be fully addressed.
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