88 MicroRNA profile of invitro bovine embryos cultured in the presence of amniotic extracellular vesicles shifts toward invivo-collected blastocysts

2020 ◽  
Vol 32 (2) ◽  
pp. 170
Author(s):  
A. Lange-Consiglio ◽  
B. Lazzari ◽  
F. Pizzi ◽  
A. Idda ◽  
F. Cremonesi ◽  
...  
Keyword(s):  
Pregnancy Rate ◽  
Extracellular Vesicles ◽  
Embryo Implantation ◽  
Principal Component ◽  
Rna Isolation ◽  
Differentially Expressed ◽  
Bovine Embryos ◽  
Total Rna ◽  
Mirna Profiling ◽  
Genomic Study

The absence of maternal-embryo signals could be an important cause of the poor pregnancy rates of invitro-produced embryos, compared with those collected invivo. In the context of paracrine communication, co-culture of embryo with amniotic-derived extracellular vesicles (EVs) improved their quality compared with control (CTR) (Perrini and Lange Consiglio 2018 Reprod. Fertil. Dev. 30, 658-671), and after cryopreservation, provided higher invitro embryo hatching and recipient pregnancy rate (Lange-Consiglio et al. 2019 Reprod. Fertil. Dev. 31, 155). After these results, the aim of this study was to evaluate microRNA (miRNA) profiling of invitro-produced blastocysts with or without EV supplementation, using invivo-produced blastocysts as CTR. Invitro embryos were produced based on our protocol (Perrini and Lange Consiglio 2018 Reprod. Fertil. Dev. 30, 658-671) with or without 100×106 EVsmL−1 in synthetic oviductal fluid with amino acids (SOFaa) on Day 5 post-fertilisation (Perrini and Lange Consiglio 2018 Reprod. Fertil. Dev. 30, 658-671). Grade 1 blastocysts (B7) were immediately snap frozen in liquid nitrogen for genomic study. These embryos were obtained from three replicates. Invivo embryos were obtained from three cows superovulated by Folltropin and inseminated by the same cryopreserved semen. After flushing, only B7 were snap frozen for genomic study. Samples for RNA isolation were obtained from 3 pools of 10 embryos each for each condition (vivo, vitro-CTR, and vitro+EVs). Total RNA was isolated by a NucleoSpin1 miRNA kit. Concentration and quality of RNA were determined by an Agilent 2100 Bioanalyzer. Libraries were prepared using TruSeq Small RNA Library Preparation kits (Illumina). Differential expression analyses between samples were run with the Bioconductor edgeR package (false discovery rate<0.05). MicroRNA cluster analysis was performed with Genesis. The average quantity of total RNA extracted from each pool was 3.5ng. Our results show that the miRNAs identified were 1.74E5, 2.3E5, and 3.6E5 for vivo, vitro-CTR, and vitro+EVs, respectively. Principal component analysis calculated on differentially expressed miRNAs showed a separation of the three groups with a distinctive miRNA trait. The miRNAs differentially expressed among three comparisons (vivo vs. vitro-CTR, vivo vs. vitro+EVs, and vitro-CTR vs. vitro+EVs) were 20, 15, and 2, respectively. Principal component 1, which explains 62.4% of the variance, clearly separates invivo- and invitro-produced embryos even if EV addition seems to ameliorate the effect of invitro production, and this agrees with the embryo quality and pregnancy rate after EV supplementation (Perrini and Lange Consiglio 2018 Reprod. Fertil. Dev. 30, 658-671; Lange-Consiglio et al. 2019 Reprod. Fertil. Dev. 31, 155). Indeed, vitro-CTR and vitro+EVs embryos differ significantly for two miRNAs (miR-130a, miR-181b) that are found to be higher in our vitro-CTR embryos compared with vitro+EV ones. The miR-181b was also found to be higher in degenerate bovine embryos compared with good blastocysts (Kropp et al. 2014 Front. Genetics 24, 91). In conclusion, this is the first study reporting the complete miRNA profiling of invitro blastocysts compared with those obtained invivo. The addition of EVs during invitro production seems to influence the expression of specific miRNAs involved in the success of embryo implantation.

177 GENE EXPRESSION PROFILES OF IN VITRO- AND IN VIVO-DERIVED BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 190
Author(s):  
D. Aktoprakligil Aksu ◽  
C. Agca ◽  
S. Aksu ◽  
T. Akkoc ◽  
A. Tas Caputcu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Functional Annotation ◽  
Differentially Expressed ◽  
Annotation Tool ◽  
Bovine Embryos ◽  
University Of Missouri ◽  
Total Rna ◽  
David Functional Annotation Tool

Microarray technology is one of the most powerful tools for gene expression profiling in animal sciences. The objectives of this study were to determine the effect of vitrification on gene expression in in vitro- and in vivo-derived bovine embryos, and to identify differential mRNA expression patterns between embryos produced by in vivo v. in vitro conditions. Three pools of in vivo- and in vitro-derived blastocyst-stage embryos were used for microarray analysis. Total RNA was isolated using the PicoPure RNA Isolation Kit (Arcturus Bioscience, Mountain View, CA). Bovine ovarian tissue total RNA was used as the reference. Total RNA samples were amplified using an Ovation® Pico WTA System (NuGEN Technologies, San Carlos, CA). The bovine 16 846-member microarrays spotted with 70-mer oligonucleotides were purchased from the Bovine Genomics Laboratory, University of Missouri. Amplified cDNA samples were labeled with Alexa Fluor 647 and 546 dyes (Molecular Probes, Eugene, OR), respectively. Combined, labeled samples were dried and resuspended in hybridization buffer containing 50% formamide (vol/vol), 5× SSC, and 0.1% sodium dodecyl sulfate (wt/vol). After denaturation and cooling, cDNA was applied onto a microarray slide. Microarrays were hybridized overnight at 42°C. Following hybridization, the slides were washed with different stringency buffers and water. After drying by centrifugation, the arrays were scanned on a GenePix 4000B scanner (Axon Instruments, Union City, CA). GenePix Pro4.1 software was used for griding and analysis of spot intensities. Good-quality spots were analyzed using the GeneSpring 7.3 software (Agilent Technologies, Inc., CA, Santa Clara, CA). The data were normalized per spot and per array by Lowess normalization. When comparing two treatments, the Welch t-test with Benjamini and Hochberg multiple testing correction was performed to determine the differentially expressed genes between embryo groups. Microarray experiments were performed in 3 biological and 2 technical replicates for all embryo samples. Differentially expressed genes between all embryo groups were identified. The DAVID Functional Annotation Tool was used to analyze the genes that were differentially expressed. The DAVID Functional Annotation Tool determined the co-occurrence probability and provided gene-GO term enrichment analysis to highlight the most relevant GO terms associated with a given gene list. Differentially expressed Kyoto Encyclopedia of Genes and Genomes pathways are as follows: Ribosome, oxidative phosphorylation, spliceosome, and oocyte meiosis were significantly upregulated in the fresh embryos, whereas sphingolipid and purine metabolism was the upregulated in the vitrified in vitro-derived embryos. Gene expression was very similar between fresh and vitrified in vivo-derived, as opposed to in vitro-derived, embryos. This study was funded by the TUBITAK (Project no. KAMAG107G027) and startup funds to Yuksel Agca at the University of Missouri.

scholarly journals Evaluation of Bovine Embryo Biopsy Techniques according to Their Ability to Preserve Embryo Viability

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
M. Cenariu ◽  
E. Pall ◽  
C. Cernea ◽  
I. Groza
Keyword(s):  
Pregnancy Rate ◽  
Genetic Diagnosis ◽  
Needle Aspiration ◽  
Embryo Biopsy ◽  
Embryo Cryopreservation ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Embryo Viability ◽  
Biopsy Methods ◽  
Biopsy Method

The purpose of this research was to evaluate three embryo biopsy techniques used for preimplantation genetic diagnosis (PGD) in cattle and to recommend the least invasive one for current use, especially when PGD is followed by embryo cryopreservation. Three hundred bovine embryos were biopsied by either one of the needle, aspiration or microblade method, and then checked for viability by freezing/thawing and transplantation to recipient cows. The number of pregnancies obtained after the transfer of biopsied frozen/thawed embryos was assessed 30 days later using ultrasounds. The results were significantly different between the three biopsy methods: the pregnancy rate was of 57% in cows that received embryos biopsied by needle, 43% in cows that received embryos biopsied by aspiration, and 31% in cows that received embryos biopsied by microblade. Choosing an adequate biopsy method is therefore of great importance in embryos that will undergo subsequent cryopreservation, as it significantly influences their viability after thawing.

scholarly journals Characterization of an Ex Vivo Equine Endometrial Tissue Culture Model Using Next-Generation RNA-Sequencing Technology

Animals ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1995
Author(s):  
Maithê R. Monteiro de Barros ◽  
Mina C. G. Davies-Morel ◽  
Luis A. J. Mur ◽  
Christopher J. Creevey ◽  
Roger H. Alison ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Ex Vivo ◽  
Prostaglandin F2α ◽  
Principal Component ◽  
Differentially Expressed ◽  
Temporal Window ◽  
Cellular Processes ◽  
Persistent Inflammation ◽  
Uterine Inflammation ◽  
Tissue Culture Model

Persistent mating-induced endometritis is a major cause of poor fertility rates in the mare. Endometritis can be investigated using an ex vivo equine endometrial explant system which measures uterine inflammation using prostaglandin F2α as a biomarker. However, this model has yet to undergo a wide-ranging assessment through transcriptomics. In this study, we assessed the transcriptomes of cultured endometrial explants and the optimal temporal window for their use. Endometrium harvested immediately post-mortem from native pony mares (n = 8) were sampled (0 h) and tissue explants were cultured for 24, 48 and 72 h. Tissues were stored in RNALater, total RNA was extracted and sequenced. Differentially expressed genes (DEGs) were defined using DESeq2 (R/Bioconductor). Principal component analysis indicated that the greatest changes in expression occurred in the first 24 h of culture when compared to autologous biopsies at 0 h. Fewer DEGs were seen between 24 and 48 h of culture suggesting the system was more stable than during the first 24 h. No genes were differentially expressed between 48 and 72 h but the low number of background gene expression suggested that explant viability was compromised after 48 h. ESR1, MMP9, PTGS2, PMAIP1, TNF, GADD45B and SELE genes were used as biomarkers of endometrial function, cell death and inflammation across tissue culture timepoints. STRING assessments of gene ontology suggested that DEGs between 24 and 48 h were linked to inflammation, immune system, cellular processes, environmental information processing and signal transduction, with an upregulation of most biomarker genes at 24 h. Taken together our observations indicated that 24–48 h is the optimal temporal window when the explant model can be used, as explants restore microcirculation, perform wound healing and tackle inflammation during this period. This key observation will facilitate the appropriate use of this as a model for further research into the equine endometrium and potentially the progression of mating-induced endometritis to persistent inflammation between 24 and 48 h.

scholarly journals Detection of Prostate Cancer via IR Spectroscopic Analysis of Urinary Extracellular Vesicles: A Pilot Study

Membranes ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 591
Author(s):  
Xin-Le Yap ◽  
Bayden Wood ◽  
Teng-Aik Ong ◽  
Jasmine Lim ◽  
Bey-Hing Goh ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Extracellular Vesicles ◽  
Cancer Progression ◽  
Prostate Cancer Screening ◽  
Principal Component ◽  
Urine Samples ◽  
Cancer Type ◽  
Marker Proteins ◽  
Novel Strategy ◽  
Ir Spectroscopic

Extracellular vesicles (EVs) are membranous nanoparticles naturally released from living cells which can be found in all types of body fluids. Recent studies found that cancer cells secreted EVs containing the unique set of biomolecules, which give rise to a distinctive absorbance spectrum representing its cancer type. In this study, we aimed to detect the medium EVs (200–300 nm) from the urine of prostate cancer patients using Fourier transform infrared (FTIR) spectroscopy and determine their association with cancer progression. EVs extracted from 53 urine samples from patients suspected of prostate cancer were analyzed and their FTIR spectra were preprocessed for analysis. Characterization of morphology, particle size and marker proteins confirmed that EVs were successfully isolated from urine samples. Principal component analysis (PCA) of the EV’s spectra showed the model could discriminate prostate cancer with a sensitivity of 59% and a specificity of 81%. The area under curve (AUC) of FTIR PCA model for prostate cancer detection in the cases with 4–20 ng/mL PSA was 0.7, while the AUC for PSA alone was 0.437, suggesting the analysis of urinary EVs described in this study may offer a novel strategy for the development of a noninvasive additional test for prostate cancer screening.

scholarly journals Comprehensive Comparison of Amnion Stromal Cells and Chorion Stromal Cells by RNA-Seq

2021 ◽  
Vol 22 (4) ◽  
pp. 1901
Author(s):  
Brielle Jones ◽  
Chaoyang Li ◽  
Min Sung Park ◽  
Anne Lerch ◽  
Vimal Jacob ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stromal Cells ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Principal Component ◽  
Differentially Expressed ◽  
Inflammatory Factor ◽  
Next Generation Sequencing Technology ◽  
Angiogenic Potential ◽  
Anti Inflammatory

Mesenchymal stromal cells derived from the fetal placenta, composed of an amnion membrane, chorion membrane, and umbilical cord, have emerged as promising sources for regenerative medicine. Here, we used next-generation sequencing technology to comprehensively compare amniotic stromal cells (ASCs) with chorionic stromal cells (CSCs) at the molecular and signaling levels. Principal component analysis showed a clear dichotomy of gene expression profiles between ASCs and CSCs. Unsupervised hierarchical clustering confirmed that the biological repeats of ASCs and CSCs were able to respectively group together. Supervised analysis identified differentially expressed genes, such as LMO3, HOXA11, and HOXA13, and differentially expressed isoforms, such as CXCL6 and HGF. Gene Ontology (GO) analysis showed that the GO terms of the extracellular matrix, angiogenesis, and cell adhesion were significantly enriched in CSCs. We further explored the factors associated with inflammation and angiogenesis using a multiplex assay. In comparison with ASCs, CSCs secreted higher levels of angiogenic factors, including angiogenin, VEGFA, HGF, and bFGF. The results of a tube formation assay proved that CSCs exhibited a strong angiogenic function. However, ASCs secreted two-fold more of an anti-inflammatory factor, TSG-6, than CSCs. In conclusion, our study demonstrated the differential gene expression patterns between ASCs and CSCs. CSCs have superior angiogenic potential, whereas ASCs exhibit increased anti-inflammatory properties.

scholarly journals Natural Cycle Results in Lower Implantation Failure than Ovarian Stimulation in Advanced-Age Poor Responders Undergoing IVF: Fertility Outcomes from 585 Patients

2021 ◽  
Author(s):  
Maria Paola De Marco ◽  
Giulia Montanari ◽  
Ilary Ruscito ◽  
Annalise Giallonardo ◽  
Filippo Maria Ubaldi ◽  
...  
Keyword(s):  
Pregnancy Rate ◽  
Ovarian Stimulation ◽  
Embryo Implantation ◽  
Implantation Rate ◽  
Poor Responder ◽  
Advanced Age ◽  
Poor Responders ◽  
Natural Cycle ◽  
Significant Difference ◽  
Over 40 Years

AbstractTo compare pregnancy rate and implantation rate in poor responder women, aged over 40 years, who underwent natural cycle versus conventional ovarian stimulation. This is a retrospective single-center cohort study conducted at the GENERA IVF program, Rome, Italy, between September 2012 and December 2018, including only poor responder patients, according to Bologna criteria, of advanced age, who underwent IVF treatment through Natural Cycle or conventional ovarian stimulation. Between September 2012 and December 2018, 585 patients were included within the study. Two hundred thirty patients underwent natural cycle and 355 underwent conventional ovarian stimulation. In natural cycle group, both pregnancy rate per cycle (6.25 vs 12.89%, respectively, p = 0.0001) and pregnancy rate per patient101 with at least one embryo-transfer (18.85 vs 28.11% respectively, p = 0.025) resulted significant reduced. Pregnancy rate per patient managed with conventional ovarian stimulation resulted not significantly different compared with natural cycle (19.72 vs 15.65% respectively, p = 0.228), but embryo implantation rate was significantly higher in patients who underwent natural cycle rather than patient subjected to conventional ovarian stimulation (13 vs 8.28% respectively, p = 0.0468). No significant difference could be detected among the two groups in terms of abortion rate (p = 0.2915) or live birth pregnancy (p = 0.2281). Natural cycle seems to be a valid treatment in patients over 40 years and with a low ovarian reserve, as an alternative to conventional ovarian stimulation.

scholarly journals Multiplexed profiling of single-cell extracellular vesicles secretion

2019 ◽  
Vol 116 (13) ◽  
pp. 5979-5984 ◽  
Author(s):  
Yahui Ji ◽  
Dongyuan Qi ◽  
Linmei Li ◽  
Haoran Su ◽  
Xiaojie Li ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Lines ◽  
Extracellular Vesicles ◽  
Single Cell Analysis ◽  
Comprehensive Evaluation ◽  
Single Cells ◽  
Deep Understanding ◽  
Principal Component ◽  
Cell Heterogeneity ◽  
Indispensable Tool

Extracellular vesicles (EVs) are important intercellular mediators regulating health and diseases. Conventional methods for EV surface marker profiling, which was based on population measurements, masked the cell-to-cell heterogeneity in the quantity and phenotypes of EV secretion. Herein, by using spatially patterned antibody barcodes, we realized multiplexed profiling of single-cell EV secretion from more than 1,000 single cells simultaneously. Applying this platform to profile human oral squamous cell carcinoma (OSCC) cell lines led to a deep understanding of previously undifferentiated single-cell heterogeneity underlying EV secretion. Notably, we observed that the decrement of certain EV phenotypes (e.g.,CD63+EV) was associated with the invasive feature of both OSCC cell lines and primary OSCC cells. We also realized multiplexed detection of EV secretion and cytokines secretion simultaneously from the same single cells to investigate the multidimensional spectrum of cellular communications, from which we resolved tiered functional subgroups with distinct secretion profiles by visualized clustering and principal component analysis. In particular, we found that different cell subgroups dominated EV secretion and cytokine secretion. The technology introduced here enables a comprehensive evaluation of EV secretion heterogeneity at single-cell level, which may become an indispensable tool to complement current single-cell analysis and EV research.

scholarly journals Comparative Transcriptomic Analysis of Embryo Implantation in Mice and Rats

2018 ◽  
Vol 50 (2) ◽  
pp. 668-678 ◽  
Author(s):  
Wen-Qian Zhang ◽  
Miao Zhao ◽  
Ming-Yu Huang ◽  
Ji-Long Liu
Keyword(s):  
Differentially Expressed Genes ◽  
Molecular Mechanisms ◽  
Embryo Implantation ◽  
Differentially Expressed ◽  
Rat Uterus ◽  
Mouse Uterus ◽  
Pathway Network ◽  
Implantation Sites ◽  
High Selection ◽  
Transcriptomic Changes

Background/Aims: Embryo implantation is an essential process for eutherian pregnancy, but this process varies across eutherians. The genomic mechanisms that led to the emergence and diversification of embryo implantation are largely unknown. Methods: In this study, we analyzed transcriptomic changes during embryo implantation in mice and rats by using RNA-seq. Bioinformatics and evolutionary analyses were performed to characterize implantation-associated genes in these two species. Results: We identified a total of 518 differentially expressed genes in mouse uterus during implantation, of which 253 genes were up-regulated and 265 genes were down-regulated at the implantation sites compared with the inter-implantation sites. In rat uterus, there were 374 differentially expressed genes, of which 284 genes were up-regulated and 90 genes were down-regulated. A cross-species comparison revealed that 92 up-regulated genes and 20 down-regulated genes were shared. The differences and similarities between mice and rats were investigated further at the gene ontology, pathway, network, and causal transcription factor levels. Additionally, we found that embryo implantation might have evolved through the recruitment of ancient genes into uterine expression. The evolutionary rates of the differentially expressed genes in mouse and rat uterus were significantly lower than those of the non-changed genes, indicating that implantation-related genes are evolutionary conserved due to high selection pressure. Conclusion: Our study provides insights into the molecular mechanisms involved in the evolution of embryo implantation.

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