Small extracellular vesicles in human uterine fluid
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Uterine Lavage, Endometrium
SUBMITTER: 
david greening
LAB HEAD: David Greening
PROVIDER: PXD020975 | Pride | 2021-03-18
REPOSITORIES: Pride
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Project description:Successful embryo implantation into a receptive endometrium requires mutual endometrial-embryo communication. Recently, the function of extracellular vehicles (EVs) in cell-to-cell interaction in embryo-maternal interactions has been investigated. We explored isolated endometrial derived EVs, using RL95-2 cells as a model of a receptive endometrium, influenced by menstrual cycle hormones estrogen (E2; proliferative phase) progesterone (P4; secretory phase) and estrogen plus progesterone (E2P4; the receptive phase). EV sized particles were isolated by differential centrifugation and size exclusion chromatography. Nanoparticle tracking analysis was used to examine the different concentration and size of particles and EV proteomic analysis per-formed using shotgun label-free mass spectrometry. Our results showed that although endome-trial derived EVs were secreted in numbers independent of hormonal stimulation, EVs sizes were statistically modified by it. Proteomics analysis showed that hormone treatment changes affect the endometrial EVs proteome, with proteins enhanced within the EV E2P4 group shown to be in-volved in different processes such as embryo implantation, endometrial receptivity, and embryo development, supporting the concept of a communication system between the embryo and the maternal endometrium via EVs.
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Project description:Improving embryo implantation rates has become one of the greatest challenges in assisted reproduction techniques. Implantation process requires a synchrony between the development of the embryo and the endometrium, but also, an adequate embryo-endometrial crosstalk. MicroRNAs (miRNAs) and extracellular vesicles (EVs) from endometrial fluid (EF) have been described to be mediators of this communication. Therefore, the molecular analysis of the miRNA content of EF could provide a non-invasive method to recognize an implantative endometrium and consequently improve the implantation rates. In this work, we optimized EF sample collection procedure and performed a comparative analysis of five different methodologies to enrich miRNA content of EF with the purpose of identifying a simple, sensitive, reproducible and non-invasive method that allow the quick identification of an implantative endometrium. Then, we applied the selected methodology in a set of real samples with different implantation outcome. Our results showed that EVs enrichment protocols permit to detect a higher number of miRNAs. In addition, we obtained two predictive models based on three miRNAs that allow us to differentiate between an implantative and non-implantative endometrium. Model 1 (Area under the curve (AUC)=0.93; p-value=0.0033): hsa-miR-200b-3p, hsa-miR-24-3p and hsa-miR-148b-3p. Model 2 (AUC=0.92; p-value=0.0002): hsa-miR-200b-3p, hsa-miR-24-3p and hsa-miR-99b-5p. This work supports the feasibility to analyze EVs and EVs-associated miRNAs from a small volume of EF sample and the development of EV-miRNAs based assays as low-invasive tools for the detection of an implantative endometrium.
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Project description:To unbiasedly and systematically characterize endometrial transformation across the human menstrual cycle in preparation for embryo implantation, we analyzed the transcriptomic transformation of human endometrium at single cell resolution, dissecting multidimensional cellular heterogeneity of the tissue across the entire natural menstrual cycle.
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Project description:Synchronized cross talk between embryo and endometrium during pre-implantation period is critical for establishment of pregnancy. Extracellular vesicels(EVs) of both embryo and endometrial origin are known to be integral in regulating embryo maternal communication during this time. However, exact molecular signalling pathways or factors that regulate the embryo endometrial communication during pre-conception period remains elusive. Here in this study extracellular vesicles from trophoblast cells were shown to modulate endometrial epithelial cell secretome in favour of embryo implantation and embryo development.
2023-10-24 | PXD040311 | Pride
Project description:Extracellular vesicles (EVs) are important mediators of embryo attachment and outgrowth critical for successful implantation. While EVs have garnered immense interest in their therapeutic potential in assisted reproductive technology by improving implantation success, their large-scale generation remains a major challenge. Here, we report a rapid and scalable production of nanovesicles (NVs) from human trophectoderm cells (hTSCs) via membrane extrusion; these NVs can be generated in approximately 6 hours with a 20-fold higher yield than EVs isolated from the same number of cells. NVs display similar biophysical traits (morphologically intact, spherical, 90-130 nm) to EVs, and are laden with hallmark players of implantation (ITGA2/V, ITGB1, PRDX1, SOD2, and MFGE8) that include cell-matrix adhesion and extracellular matrix organisation proteins. Functionally, NVs are readily taken up by low-receptive endometrial HEC1A cells and reprogram their proteome towards a receptive phenotype that support hTSC spheroid attachment. Moreover, a single dose treatment of NVs significantly enhanced adhesion and spreading of mouse embryo trophoblast on fibronectin matrix. We show functional potential of NVs in enhancing embryo implantation and highlight their rapid and scalable generation, amenable to clinical utility.
2023-02-20 | PXD038856 | Pride
Project description:Study question: Could extracellular vesicles (EVs) secreted by aneuploid embryos a) serve for development of RNA biomarkers for preimplantation genetic testing for aneuploidy (PGT-A) and b) elicit a relevant transcriptomic response in decidualised primary endometrial stromal cells (dESCs)? Summary answer: Aneuploid embryo EVs a) contain genes PPM1J, LINC00561, ANKRD34C and TMED10 in differential abundance from euploid EVs and b) induce up-regulation of MUC1 transcript in dESCs. What is known already: PGT-A is popular as it is thought to facilitate transfer of euploid embryos to increase implantation probability but the technology is controversial, as it requires an invasive embryo biopsy with an elusive long-term biosafety. The development of non-invasive methods to screen out aneuploid embryos is paramount. It is also critical to decode the embryo-endometrial dialog underlying implantation failure. We have previously reported that IVF embryos secrete EVs that can be internalised by ESCs, conceptualising that successful implantation to the endometrium is facilitated by EVs, which may additionally serve as biomarkers of ploidy status. Study design, size, duration: Embryos destined for biopsy on days 5-7 for PGT-A were grown under standard conditions. Spent media (30μl) were collected from euploid (n=175) and aneuploid (n=140)embryos at cleavage (days 1-3) stage and from euploid (n=187) and aneuploid (n=142) embryos at blastocyst (days 3-5) stage. Media samples from n=35 cleavage embryos were pooled in order to obtain five euploid and four aneuploid pools. Similarly, media samples from blastocyst were pooled to create one euploid and one aneuploid pool. ESCs were obtained from five women undergoing diagnostic laparoscopy. Participants/materials, setting, methods: EVs were isolated from pools of media derived from euploid and aneuploid day 3 embryos with differential centrifugation and EV-RNA sequencing was performed following the SMARTer Stranded Total RNA-Seq approach. ESCs were decidualised (E2:10nM, P4:1uM, cAMP:0.5 mM twice every 48 hours) and treated for 24 hours with 50 ng/ml euploid or aneuploid EVs extracted from blastocyst media. RNA sequencing was performed on ESCs following the Illumina Truseq RNAseq protocol. Main results and the role of chance: Aneuploid cleavage stage embryos secreted EVs that were less abundant in RNA fragments originating from the genes PPM1J (log2fc=-5.13, p=0.011), LINC00561 (log2fc=-7.87, p=0.010) and ANKRD34C (log2fc=-7.30, p=0.017) and more abundant in TMED10 (log2fc=1.63 p=0.025) compared to EVs from euploid embryos. Decidualisation per se induced downregulation of MUC1 (log2FC=-0.54, p=0.0028) in ESCs as prerequisite for the establishment of receptive endometrium. The expression of MUC1 transcript in decidualised ESCs was significantly increased following treatment with aneuploid compared to euploid embryo-secreted EVs (log2FC=0.85, p=0.0201). Limitations, reasons for caution: The findings of the study may require validation utilising a second cohort of EVs samples. Wider implications of the findings: The discovery that the RNA cargo of EVs secreted from aneuploid cleavage stage embryos is diverse from that of euploid embryos potentiates the development of non-invasive methodology for PGT-A. The upregulation of MUC1 in dESCs following aneuploid embryo EV treatment proposes a new mechanism underlying implantation failure. Funding: The study was supported by a MSCA fellowship awarded to SM by the European Commission (CERVINO grant agreement ID: 79620) and by a BIRTH research grant from Theramex HQ UK Ltd
2024-04-03 | GSE234338 | GEO
Project description:Purpose: Extracellular vescicles (EVs) of uterine origin have recently been identified, but they are not comprised by current diagnostics on endometrial receptivity. In order to test the possibility to use EVs as biomarker reservoirs for non-invasive monitoring of the endometrial status, we defined two experimental designs: in step a) we aimed at assessing the concordance between the transcriptome of endometrial tissue and uterine fluid-derived EVs (UF-EVs); in step b) we aimed at unraveling the transcriptomic profile of UF-EVs released during the receptive phase and associated with successful implantation Methods: RNAseq analysis was performed on total RNA isolated from a) endometrial biopsies and UF-EVs concomitantly collected from regularly cycling women; b) UF-EVs collected in the pre-receptive (LH+2) versus the receptive (LH+7) phase of provenly fertile women and in the receptive phase (LH+7) of women undergoing assisted reproduction and transfer of an euploid blastocyst. In the latter group, the outcomes of the following embryo transfer attempt were recorded, in order to compare the transcriptomic profile of women with successful versus failed implantation. Differential Gene Expression (DGE) and Gene Set Enrichment (GSEA) analyses were performed. Results: In the first experimental design, a highly significant correlation was found between the transcriptional profiles of endometrial tissue biopsies and corresponding UF-EVs (r=0.71 p<0.001; ρ=0.65 p<0.001). In the second experimental design, using a large group of 73 samples, 16,777 genes were overall found to be expressed in UF-EVs. Of these, DGE analysis showed that 942 were up-regulated and 1,305 down-regulated during the receptive phase in women with proven fertility and 97 were up-regulated and 64 down-regulated during the receptive phase of women achieving successful implantation versus women failing to achieve pregnancy in the subsequent cycle. 41 genes had a statistically significant different expression in both comparisons. We additionally tested wether transcripts used by current endometrial receptivity tests based on endometrial biopsies (Endometrial Receptivity Assay, ERA test) could also be detected in UF-EVs: out of 238 ERA test gene data-set, GSEA highlighted 219 genes with similar transcriptional profile in UF-EVs. We then performed another comparison adding the list of genes consisting of the ‘metasignature’ transcriptome derived from the published meta-analysis of endometrial receptivity associated transcripts, comprising 57 genes. This last intersection identified 38 genes expressed in UF-EVs that i) show differential expression with the same transcriptional profile (same trends) in both EVs and ERA test assay; ii) belong to the leading edge of genes with the most significant enrichment score in our analysis; iii) are identified as receptivity-associated genes by the current meta-analysis on endometrial transcriptome. Conclusions: This is the first study showing the capacity of UF-EVs to reflect the endometrial tissue transcriptional status and to establish a signature of genes expressed in UF-EVs that might serve as putative biomarkers for non-invasive endometrial receptivity tests.
2021-07-01 | GSE158958 | GEO
Project description:Mechanisms of implantation such as determination of the attachment pole, fetal-maternal communication, as well as underlying causes of implantation failure are largely unexplored. Here we performed single-cell RNA sequencing (scRNA-seq) on peri-implantation embryos from both humans and mice to explore trophectoderm development and embryo–endometrium crosstalk. We found that the transcriptomes of polar and mural trophectoderm (TE) diverged after embryos hatched from the zona pellucida in both species, with polar TE being more mature than mural TE. The implantation poles show similarities in cell cycle activities, as well as in expression of genes critical for implantation and placentation. Embryos that either fail to attach in vitro or fail to implant in vivo, show abnormalities in pathways related to energy production, protein metabolism and 18S rRNA m6A methylation. These findings uncover the gene expression characteristics of humans and mice TE differentiation during the peri-implantation period and provide new insights into embryo implantation.
2022-08-01 | GSE133200 | GEO