scholarly journals Fluorescent-dependent Comparative Ct Method for qPCR Gene Expression Analysis in IVF Clinical Pre-Implantation Embryonic Testing

2021 ◽  
Author(s):  
Arnav Lal ◽  
William E Roudebush ◽  
Monica Mainigi ◽  
Renee J Chosed
Keyword(s):  
Gene Expression ◽  
Stochastic Systems ◽  
Expression Patterns ◽  
Control Group ◽  
Specific Gene ◽  
Gene Target ◽  
Vitro Fertilization ◽  
Modified Method ◽  
Fluid Analysis

Abstract The use of quantitative PCR (qPCR) and other PCR-based methods in the field of human in-vitro fertilization (IVF) blastocoel fluid analysis can potentially be utilized for assisting clinicians in embryo selection based on specific gene expression patterns. Since typical Comparative Ct analysis utilizes one threshold for runs per gene target and requires an inherent control group, this method is inadequate for analysis of small stochastic systems, such as embryonic-derived fluid. We mathematically demonstrate analytical modifications upon the Comparative Ct qPCR workflow to incorporate a variable fluorescence threshold (utilizing only the parameters defined in the Comparative Ct method), and subsequently demonstrate the typical workflow in which this modified method can successfully quantifiably analyze embryonic blastocoel fluid qPCR analysis.

scholarly journals 106 GENOMIC IMPRINTING OF IGF2R IN TISSUES OF BOVINE FETUSES GENERATED BY ARTIFICIAL INSEMINATION OR IN VITRO FERTILIZATION

2005 ◽  
Vol 17 (2) ◽  
pp. 204 ◽  
Author(s):  
S. Hiendleder ◽  
D. Bebbere ◽  
S. Bauersachs ◽  
M. Stojkovic ◽  
H. Wenigerkind ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Artificial Insemination ◽  
Expression Patterns ◽  
Allelic Expression ◽  
Paternal Allele ◽  
Specific Gene ◽  
Vitro Fertilization ◽  
Bovine Fetuses ◽  
Parent Of Origin

The insulin-like growth factor 2 receptor gene (IGF2R) is involved in fetal growth regulation. A study in sheep associated fetal overgrowth after in vitro embryo culture with abnormal DNA methylation and expression of IGF2R (Young et al. 2001 Nat. Genet. 27, 153–154). This suggested that abnormal IGF2R imprinting is a major cause of fetal overgrowth. To test this hypothesis in bovine fetuses, we developed a microsatellite marker for IGF2R from cDNA sequence data and screened 45 Day-80 fetuses generated in vivo, by artificial insemination (AI), or in vitro, by in vitro fertilization (IVF) procedures, for parent-of-origin-specific gene expression. A total of 17 fetuses were heterozygous, but available parental DNA samples showed that only 12 (8 AI, 4 IVF) allowed unambiguous discrimination of parental alleles. Parent-of-origin-specific allelic expression patterns indicated that bovine IGF2R was expressed predominantly from the maternal allele and thus imprinted in fetal heart, kidney, liver, lung, muscle, and cotyledon tissue. However, the relative amount of expression from the paternal allele was tissue-specific and ranged from 6.4 ± 0.8% in skeletal muscle up to 27.4 ± 0.9% in cotyledon (SPSS or 11.5, ANOVA, P < 0.001). Tissues that originated from the same germ layer showed similar allelic expression ratios whereas significantly different expression ratios (P < 0.05) were observed between tissues originating from different germ layers. Contrary to expectations from sheep data, there was no evidence for gross abnormalities in IGF2R imprinting in tissues from overgrown (n = 2) or normal sized (n = 2) IVF fetuses. However, relative paternal expression levels in several tissues showed significant relationships (P < 0.05–0.001) with growth parameters and pointed to subtle changes in paternal IGF2R expression in overgrown IVF fetuses. We thank W. Scholz and M. Weppert for excellent technical assistance.

134 TRANSCRIPTOME PROFILE OF BOVINE BLASTOCYSTS DERIVED FROM ALTERNATIVE IN VIVO AND IN VITRO CULTURE CONDITIONS AT SPECIFIC PHASES OF EARLY EMBRYONIC DEVELOPMENT

2012 ◽  
Vol 24 (1) ◽  
pp. 179 ◽  
Author(s):  
A. Gad ◽  
U. Besenfelder ◽  
V. Havlicek ◽  
M. Hölker ◽  
M. U. Cinar ◽  
...  
Keyword(s):  
Embryonic Development ◽  
In Vitro Culture ◽  
In Vitro Maturation ◽  
Expression Patterns ◽  
Culture Conditions ◽  
Control Group ◽  
Transcriptome Profile ◽  
Vitro Fertilization

An understanding of gene expression patterns due to altered environmental conditions during different time points of the pre-implantation period would improve our knowledge on regulation of embryonic development and improve success of embryo culture. The aim of this study was to examine the effect of alternative in vivo and in vitro culture conditions at specific phases of early embryonic development on transcriptome profile of bovine blastocysts. Using nonsurgical endoscopic oviducal transfer technology, 5 different blastocyst groups were produced. The first 2 groups were matured in vitro and then either transferred after maturation or after in vitro fertilization to synchronized recipients. The other 3 groups were matured, fertilized and cultured in vitro until 4-cell, 16-cell and morula stage before transfer. Blastocysts from each group were collected by uterine flushing at Day 7 and pooled in groups of 10. Complete in vitro (IVP) and in vivo blastocysts were produced and used as controls. A unique custom microarray (Agilent) containing 42 242 oligo probes (60-mers) was used over 6 replicates of each group vs the in vivo control group to examine the transcriptome profile of blastocysts. Compared with the in vivo control group, clear dramatic shifts were found in the number of differentially expressed genes (DEG, fold change ≥2) at 2 different time points. The first shift occurred for blastocyst groups that were transferred after in vitro fertilization and before embryonic genome activation (EGA). The second shift occurred for blastocyst groups that were transferred after EGA, as well as for the IVP group. Ontological classification of DEG showed that the more time spent under in vitro conditions, the higher the percentage of DEG involved in cell death and apoptotic processes. Moreover, lipid metabolism was the most significant process affected in the blastocysts transferred after in vitro maturation and blastocysts transferred at 16-cell stage. Most DEG involved in this process were down-regulated. Pathway analysis revealed that signalling pathways were the dominant pathways in all groups except the group that was transferred after in vitro maturation. That group showed significant down-regulation for genes involved in retinoic acid receptors activation pathways. These results showed that fertilization and EGA were the most critical developmental stages influenced by in vitro culture conditions and subsequently affect blastocyst quality, as measured in terms of gene expression patterns. Moreover, we identified molecular mechanisms and pathways that were influenced by altered culture conditions. These findings will enable the examination of strategies for modifying in vitro culture conditions at critical stages that will allow more efficient production of developmentally competent blastocysts.

Different temporal gene expression patterns for ovine pre-implantation embryos produced by parthenogenesis or in vitro fertilization

2010 ◽  
Vol 74 (5) ◽  
pp. 712-723 ◽  
Author(s):  
Daniela Bebbere ◽  
Luisa Bogliolo ◽  
Federica Ariu ◽  
Stefano Fois ◽  
Giovanni Giuseppe Leoni ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Fertilization ◽  
Expression Patterns ◽  
Gene Expression Patterns ◽  
Vitro Fertilization ◽  
Temporal Gene Expression

scholarly journals Aberrant gene expression patterns in placentomes are associated with phenotypically normal and abnormal cattle cloned by somatic cell nuclear transfer

2008 ◽  
Vol 33 (1) ◽  
pp. 65-77 ◽  
Author(s):  
Robin E. Everts ◽  
Pascale Chavatte-Palmer ◽  
Anthony Razzak ◽  
Isabelle Hue ◽  
Cheryl A. Green ◽  
...  
Keyword(s):  
Gene Expression ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Expression Patterns ◽  
Pathological Finding ◽  
Culture Methods ◽  
Vitro Fertilization ◽  
Near Term

Transcription profiling of placentomes derived from somatic cell nuclear transfer (SCNT, n = 20), in vitro fertilization (IVF, n = 9), and artificial insemination (AI, n = 9) at or near term development was performed to better understand why SCNT and IVF often result in placental defects, hydrops, and large offspring syndrome (LOS). Multivariate analysis of variance was used to distinguish the effects of SCNT, IVF, and AI on gene expression, taking into account the effects of parturition (term or preterm), sex of fetus, breed of dam, breed of fetus, and pathological finding in the offspring (hydrops, normal, or other abnormalities). Differential expression of 20 physiologically important genes was confirmed with quantitative PCR. The largest effect on placentome gene expression was attributable to whether placentas were collected at term or preterm (i.e., whether the collection was because of disease or to obtain stage-matched controls) followed by placentome source (AI, IVF, or SCNT). Gene expression in SCNT placentomes was dramatically different from AI ( n = 336 genes; 276 >2-fold) and from IVF ( n = 733 genes; 162 >2-fold) placentomes. Functional analysis of differentially expressed genes (DEG) showed that IVF has significant effects on genes associated with cellular metabolism. In contrast, DEG associated with SCNT are involved in multiple pathways, including cell cycle, cell death, and gene expression. Many DEG were shared between the gene lists for IVF and SCNT comparisons, suggesting that common pathways are affected by the embryo culture methods used for IVF and SCNT. However, the many unique gene functions and pathways affected by SCNT suggest that cloned fetuses may be starved and accumulating toxic wastes due to placental insufficiency caused by reprogramming errors. Many of these genes are candidates for hydrops and LOS.

scholarly journals Studying some Immunological and Hormonal profiles in Infertile Male

2011 ◽  
Vol 8 (1) ◽  
pp. 381-387
Author(s):  
Baghdad Science Journal
Keyword(s):  
Normal Limit ◽  
Seminal Fluid ◽  
Lower Percentage ◽  
Control Group ◽  
Infertile Male ◽  
Fluid Level ◽  
Vitro Fertilization ◽  
Fluid Analysis ◽  
Sperm Antibodies

The present study was designed to evaluate the immunological status in a sample of Iraqi males with primary infertility and them age range18-55 years, who were attending the Centre of Infertility and in vitro Fertilization (Kamal Al-Samaraie Hospital, Baghdad) during the period December 2008 – April 2009. They were divided into three groups; 40 patients with anti-sperm antibodies (ASA), 20 patients with Asthenozoospermia (AST) and 20 patients with azoospermia (AZO). In adition to20 fertile males was as control group. The parameters of evaluations were standard seminal fluid analysis, anti-sperm antibodies and anti-mitochondrial antibodies in serum, Therefore, two types of samples were collected from each subject; seminal fluid and blood. The following results were obtained: 1. There was a significant (P ? 0.05) decrease count of sperms in ASA (44.6 x 106 sperm/ml) and AST (46.9 x 106 sperm/ml) patients as compared to controls (63.2 x 106 sperm/ml) but the the result with in normal limit. 2. Serum anti-sperm antibodies were positive in 100.% of ASA patients, while in AZO patients, a much lower percentage was observed (25% for serum), and a much lower percentage was observed in controls (5% for serum but these influence were not clear). In contrast, none of the AST patients were positive ASA. These results were positively correlated with the corresponding serum and seminal fluid level, and the highest level was observed in ASA patients (107.6 U/ml). These differences were statistically significant. 3. Serum AMA showed different percentages in ASA, AST and AZO patients and controls (37.5, 25.0 and 20.0, 15.0%, respectively), but these differences were not significant. However, their serum level was significantly increased in ASA patients as compared to controls (11.9 vs. 6.5 U/ml).

164 EPIGENETIC ANALYSIS OF GENOMIC DNA IN PREPUBERAL AND ADULT BOVINE OOCYTES

2011 ◽  
Vol 23 (1) ◽  
pp. 184
Author(s):  
M. Diederich ◽  
J. Heinzmann ◽  
W. Kues ◽  
U. Baulain ◽  
T. Haaf ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Methylation Status ◽  
Control Group ◽  
Specific Gene ◽  
In Vitro Production ◽  
Adult Cattle ◽  
Developmental Potential ◽  
Bovine Oocytes

The use of oocytes obtained from prepuberal cattle shortens the generation interval by producing descendants of genetically valuable animals before achieving actual cultivation maturity. However, several studies proved that oocytes derived from prepuberal animals differ significantly from oocytes of adult animals with regard to their developmental capability and therefore reproductive potential. Epigenetic events are taken into consideration as a possible reason for this phenomenon. Particularly DNA methylation, allele specific gene expression in a parent-of-origin-specific manner (imprinting), and certain histone modifications, like acetylations, carboxylations, and phosphorylations, play an important role. This project aims to gain knowledge about the mechanisms involved in attaining of the full developmental potential of bovine oocytes. Using immature and in vitro matured oocytes of prepuberal and adult cattle, a comparative study was conducted by measuring mRNA expression of 4 developmentally relevant, but non-imprinted genes (GDF9, GLUT1, PRDX1, and ZAR1) as well as the general DNA methylation status, performed by bisulfite sequencing of 2 satellite sequences [bovine testis satellite I DNA segment 2 (BTSS2) and Bos taurus α satellite I DNA (BTS)]. After various pretreatments, immature bovine oocytes were collected from prepuberal calves [6–9 months, either left untreated (Ca1) or treated with FSH (Ca2) or FSH+IGF1 (Ca3) or FSH+IGFK (Ca4)] and adult animals [≥2nd lactation, either left untreated (Ad1) or treated with FSH (Ad2)] using the Ovum-pick-up (OPU) technique. The Ad1 group was considered the control group. First results of the qPCR analyses of immature oocytes show differences between treatment groups for GLUT1, PRDX1, and ZAR1 transcripts. Compared with Ad1, GLUT1 expression increased in Ad2 [fold change (FC) 2.2], Ca1 (FC 2.0), Ca2 (FC 1.8), and Ca3 (FC 1.4). The genes PRDX1 and ZAR1 were reduced in all groups by 0.02 to 0.07 in comparison with Ad1. The GDF9 showed generally a very low expression. The methylation analysis shows for BTSS2 and BTS significant differences before and after in vitro maturation in the groups Ad1 (BTSS2: 49.6 v. 64.9%), Ad2 (BTS: 76.7 v. 52.5%), Ca1 (BTSS2: 74.6 v. 53.3%), Ca2 (BTS: 72.8 v. 57.8%) and Ca3 (BTSS2: 60.6 v. 71.7%). Currently, the first experiment and statistical analysis are under way. The preliminary data confirm differences in gene expression between prepuberal and adult animals, and demonstrates the dependence of the methylation pattern on age and maturation status. These results contribute to a better understanding of the developmental potential of prepuberal oocytes in order to optimize their use for in vitro production of embryos. This work was supported by the H. Wilhelm Schaumann Foundation, Hamburg.

scholarly journals Stimulating Effect of a Newly Synthesized Sulfonamido-Basedgallate on Articular Chondrocytes in Vitro

2015 ◽  
Vol 37 (3) ◽  
pp. 1196-1209
Author(s):  
Zhenhui Lu ◽  
Liqin Wang ◽  
Hongmei Pan ◽  
Xiao Lin ◽  
Cuiwu Lin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transforming Growth Factor ◽  
Articular Chondrocytes ◽  
Glycogen Synthase ◽  
Control Group ◽  
Specific Gene ◽  
Cytotoxicity Test ◽  
Regulated Gene Expression ◽  
Tgf Β1

Background: The phenotype of chondrocyte is easy to be lost when expanded in vitro by a process defined “dedifferentiation”. Traditional growth factors such as transforming growth factor (TGF-β1) are effective in preventing of dedifferentiation, but high costs and loss of activity limited their use. It is of significance to find substitutes which can reduce dedifferentiation and preserve chondrocytes phenotype to ensure sufficient differentiated cells for further study. Methods: We synthesized new type of sulfonamido-based gallates named ZXHA-C and investigated its effect on primary articular chondrocytes of rats. After preliminary screening by cytotoxicity test, ZXHA-C of 1.06 × 10-8, 1.06 × 10-7 and 1.06 × 10-6M were chosen for further studies. Cell proliferation, morphology, viability, GAG synthesis and cartilage specific gene expression were detected. Also the effects of ZXHA-C on Wnt/β-catenin signaling pathway were investigated. Results: ZXHA-C could significantly promote chondrocytes growth. And it could enhance ECM synthesis by up-regulating expression levels of cartilage specific markers like aggrecan, collagen II and Sox9. Expression of collagen I which marked chondrocytes dedifferentiation was also significantly down-regulated after treated by ZXHA-C. Further exploration of the molecular mechanism indicated that ZXHA-C activated the Wnt/β-catenin signal pathway in chondrocytes, as evidenced by up-regulated gene expression of β-catenin, Wnt-4, cyclin D1 and Frizzled-2 and decreased glycogen synthase kinase 3β (GSK-3β). Among the various concentrations, ZXHA-C of 1.06 × 10-7 M showed the best performance, which was close to positive control (group with TGF-β1). Conclusion: ZXHA-C might be potential a novel agent for the maintenances of chondrocytes phenotype.

scholarly journals A Biomimetic Biphasic Scaffold Consisting of Decellularized Cartilage and Decalcified Bone Matrixes for Osteochondral Defect Repair

2021 ◽  
Vol 9 ◽  
Author(s):  
Runfeng Cao ◽  
Anqi Zhan ◽  
Zheng Ci ◽  
Cheng Wang ◽  
Yunlang She ◽  
...  
Keyword(s):  
Gene Expression ◽  
Subchondral Bone ◽  
Bone Matrix ◽  
Control Group ◽  
Specific Gene ◽  
Specific Gene Expression ◽  
Osteochondral Repair ◽  
Composition And Structure ◽  
Structural And Functional Properties

It is challenging to develop a biphasic scaffold with biomimetic compositional, structural, and functional properties to achieve concomitant repair of both superficial cartilage and subchondral bone in osteochondral defects (OCDs). This study developed a biomimsubchondraletic biphasic scaffold for OCD repair via an iterative layered lyophilization technique that controlled the composition, substrate stiffness, and pore size in each phase of the scaffold. The biphasic scaffold consisted of a superficial decellularized cartilage matrix (DCM) and underlying decalcified bone matrix (DBM) with distinct but seamlessly integrated phases that mimicked the composition and structure of osteochondral tissue, in which the DCM phase had relative low stiffness and small pores (approximately 134 μm) and the DBM phase had relative higher stiffness and larger pores (approximately 336 μm). In vitro results indicated that the biphasic scaffold was biocompatible for bone morrow stem cells (BMSCs) adhesion and proliferation, and the superficial DCM phase promoted chondrogenic differentiation of BMSCs, as indicated by the up-regulation of cartilage-specific gene expression (ACAN, Collagen II, and SOX9) and sGAG secretion; whereas the DBM phase was inducive for osteogenic differentiation of BMSCs, as indicated by the up-regulation of bone-specific gene expression (Collagen I, OCN, and RUNX2) and ALP deposition. Furthermore, compared with the untreated control group, the biphasic scaffold significantly enhanced concomitant repair of superficial cartilage and underlying subchondral bone in a rabbit OCD model, as evidenced by the ICRS macroscopic and O’Driscoll histological assessments. Our results demonstrate that the biomimetic biphasic scaffold has a good osteochondral repair effect.

scholarly journals Gene-expression profiling elucidates molecular signaling networks that can be therapeutically targeted in vestibular schwannoma

2014 ◽  
Vol 121 (6) ◽  
pp. 1434-1445 ◽  
Author(s):  
Sameer Agnihotri ◽  
Isabel Gugel ◽  
Marc Remke ◽  
Antje Bornemann ◽  
Georgios Pantazis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Cell Line ◽  
Expression Profiling ◽  
Expression Patterns ◽  
Mtor Pathway ◽  
Signaling Networks ◽  
Benign Tumors ◽  
Specific Gene

ObjectVestibular schwannomas (VS) are common benign tumors of the vestibular nerve that cause significant morbidity. The current treatment strategies for VS include surgery or radiation, with each treatment option having associated complications and side effects. The transcriptional landscape of schwannoma remains largely unknown.MethodsIn this study the authors performed gene-expression profiling of 49 schwannomas and 7 normal control vestibular nerves to identify tumor-specific gene-expression patterns. They also interrogated whether schwannomas comprise several molecular subtypes using several transcription-based clustering strategies. The authors also performed in vitro experiments testing therapeutic inhibitors of over-activated pathways in a schwannoma cell line, namely the PI3K/AKT/mTOR pathway.ResultsThe authors identified over 4000 differentially expressed genes between controls and schwannomas with network analysis, uncovering proliferation and anti-apoptotic pathways previously not implicated in VS. Furthermore, using several distinct clustering technologies, they could not reproducibly identify distinct VS subtypes or significant differences between sporadic and germline NF2–associated schwannomas, suggesting that they are highly similar entities. The authors identified overexpression of PI3K/AKT/mTOR signaling networks in their geneexpression study and evaluated this pathway for therapeutic targeting. Testing the compounds BEZ235 and PKI-587, both novel dual inhibitors of PI3K and mTOR, attenuated tumor growth in a preclinical cell line model of schwannoma (HEI-293). In vitro findings demonstrated that pharmacological inhibition of the PI3K/AKT/mTOR pathway with next-generation compounds led to decreased cell viability and increased cell death.ConclusionsThese findings implicate aberrant activation of the PI3K/AKT/mTOR pathway as a molecular mechanism of pathogenesis in VS and suggest inhibition of this pathway as a potential treatment strategy.

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