scholarly journals 82 EFFECT OF HIGH FETAL CALF SERUM CONCENTRATION IN THE GENE EXPRESSION PATTERN OF IN VITRO PRODUCED BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 155
Author(s):  
M. J. Sudano ◽  
D. M. Paschoal ◽  
E. S. Caixeta ◽  
R. R. Maziero ◽  
M. D. Guastali ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Lipid Metabolism ◽  
Expression Pattern ◽  
Expression Profiles ◽  
Rna Extraction ◽  
Gene Expression Pattern ◽  
Differentially Expressed ◽  
Bovine Embryos

Even though FCS provides energy substrates, amino acids, vitamins, growth factors, and heavy-metal chelators, its supplementation has been associated with several embryo abnormalities such as mitochondrial degeneration, metabolic deviations, excessive lipid accumulation, and decreased embryo survival after cryopreservation. The aim of the present study was to evaluate the effect of high FCS concentration in the gene expression pattern of in vitro-produced bovine embryos. Slaughterhouse ovaries were used to obtain oocytes (N = 360), which were matured and fertilized in vitro (Day 0). Presumptive zygotes were divided in 2 culture media: with low (SOFaa with 0.5% BSA and 2.5% FCS) or high (SOFaa with 0.5% BSA and 10% FCS) FCS concentration. Cleavage was evaluated on Day 3. Embryo development was evaluated after 7 days under standard culture conditions (at 38.5°C in atmosphere of 5% O2, 5% CO2, and 90% N2). The produced blastocysts were placed in PBS solution and washed five times. A single blastocyst was frozen in a minimal volume of PBS and stored at –80°C until RNA extraction. Total RNA extraction was performed using the PicoPure RNA isolation Kit (Applied Biosystems®, Foster City, CA, USA). Extracted RNA was evaluated through 2100-Bioanalyzer (Agilent Technologies®, Palo Alto, CA, USA) and DNAse treated (Qiagen®, Valencia, CA, USA). RiboAmp RNA Amplification Kit (Applied Biosystems®) was used to amplify the RNA (T7 RNA polymerase-catalysed amplification reaction). The aRNA output was evaluated through NanoDrop ND-1000 (NanoDrop Technologies®, Wilmington, DE, USA). A biotin-labelled cRNA and fragmented cRNA were obtained through 3′IVT Express Kit (Affymetrix®, Santa Clara, CA, USA) to perform the hybridization (N = 3 per group) using GeneChip Bovine Genome Array (Affymetrix®). Following hybridization, probe arrays were washed, stained, and scanned. Microarray data analysis was performed in the software FlexArray 1.6.1.1. Genes with a fold change of at least 1.5 and a probability of P < 0.05 were considered differentially expressed. The data from in vitro embryo production were analysed through the PROC GLM (SAS Institute Inc., Cary, NC, USA). Cleavage rate (81.4 ± 1.5 and 85.5 ± 1.4) and blastocyst production (41.8 ± 2.4 and 47.2 ± 2.8) were not different (P > 0.05) between low and high FCS concentrations, respectively. A total of 40 genes were differentially expressed between low and high FCS concentration. A total of 28 genes were annotated, with 37 genes up-regulated and 3 genes down-regulated by high FCS concentration. The associated network functions of gene expression, RNA damage and repair, and post-transcriptional modification; and cell-to-cell signalling and interaction were generated by Ingenuity Pathway Analysis® (Redwood City, CA, USA). Differentially expressed genes involved in carbohydrate metabolism (GAPVD1, MGAT4A), lipid metabolism (ELOVL5), cellular assembly and organisation (EZR, LRP2), and cell death and survival (DRT8) were identified. In conclusion, high FCS supplementation was associated with different expression profiles of genes regulating carbohydrate and lipid metabolism, cellular assembly and organisation, and cell death and survival. The authors acknowledge support from FAPESP and LNBio-CNPEM.

48 GLOBAL GENE EXPRESSION PATTERN OF BOS INDICUS AND BOS TAURUS VITRIFIED EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 117
Author(s):  
R. Cancian ◽  
M. Macelai ◽  
G. Tavares ◽  
R. S. Valente ◽  
E. S. Caixeta ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Cellular Response ◽  
Bos Taurus ◽  
Bos Indicus ◽  
Gene Expression Pattern ◽  
Global Gene Expression ◽  
Differentially Expressed ◽  
Canonical Pathways

The cryopreservation of in vitro-produced (IVP) bovine embryos is one of the most challenging areas of the assisted reproductive biotechnologies. The aim of the present study was to evaluate the global gene expression pattern of Bos indicus (Nellore) and Bos taurus (Simmental) IVP embryos after vitrification. Follicular aspiration was performed on Nellore (n = 14) and Simmental (n = 14) cows, and oocytes (n = 840 and 450; respectively) were submitted to in vitro maturation and in vitro fertilization. Presumptive zygotes were denuded and cultured in SOFaa with 0.5% BSA and 2.5% FCS during 7 days under standard culture conditions. Blastocysts (grade 1 and 2) were vitrified, warmed, and cultured for an additional 12 h under the same conditions. Nellore (n = 8) and Simmental (n = 8) IVP blastocysts considered viable after vitrification, with re-expanded blastocoel, were submitted to total RNA extraction (PicoPure, Arcturus, Applied Biosystems®, Foster Dity, CA, USA), DNAse I treatment (Qiagen®, Valencia, CA, USA), and amplification (RiboAmp, Applied Biosystems®). Fragmented cRNA were obtained through 3′IVT Express Kit (Affymetrix®, Santa Clara, CA, USA) to perform the hybridization using GeneChip Bovine Genome Array (Affymetrix®). Microarray data analysis was performed using the FlexArray 1.6.1.1 software. Genes with at least a 1.5-fold change and a P-value of less than 0.05 were considered differentially expressed. Of the 1278 genes differentially expressed between Bos taurus and Bos indicus vitrified embryos, 1108 were annotated, with 1193 genes up-regulated and 85 genes down-regulated in Bos taurus compared with Bos indicus IVP vitrified embryos. Differentially expressed genes were associated with the functional networks of cell cycle, cellular movement and DNA replication, recombination and repair; RNA post-transcriptional modifications; gene expression, protein synthesis; RNA damage and repair; cellular function and maintenance; and cell death and survival. The top 6 canonical pathways generated by Ingenuity Pathway Analysis® with the differentially expressed genes were ELF2 signalling, oxidative phosphorylation, tricarboxylic acid cycle, protein ubiquitination pathway, mTOR signalling, and IGF-1 signalling. In conclusion, Bos taurus IVP embryos seem to trigger different cellular response mechanisms to the vitrification stress in comparison with Bos indicus IVP embryos. Differential response is mainly represented by different expression profiles of genes regulating important canonical pathways involved in cellular response to stress that could be related with the higher post-cryopreservation survival capacity observed in Bos taurus embryos.Research was supported by FAPESP, CNPq, FAPERGS, and LNBio – National Laboratory of Biosciences/MCT.

Gene expression in the in vitro-produced preimplantation bovine embryos

Zygote ◽  
2007 ◽  
Vol 15 (4) ◽  
pp. 355-367 ◽  
Author(s):  
H. Badr ◽  
G. Bongioni ◽  
A.S.S. Abdoon ◽  
O. Kandil ◽  
R. Puglisi
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Gene Expression Pattern ◽  
Bovine Embryos ◽  
Embryonic Genome ◽  
Altered Gene ◽  
Genome Activation

SummaryRecent studies have demonstrated the relevance of a gene expression profile as a clinically important key feature determining embryo quality during the in vitro preimplantation period. Although the oocyte origin can play a crucial role in blastocyst yield, the postfertilization culture period has a profound effect in determining the blastocyst quality with particular regard to the relative abundance of many developmentally and clinically important candidate genes. During the preimplantation period, the embryo undergoes several morphogenetic developmental events including oocyte maturation, minor and major forms of embryonic genome activation and transition of transcription from maternal to embryonic control. The effect of an altered gene expression pattern on the in vitro-produced bovine embryos, particularly when cultured under suboptimal conditions, was reflected by the occurrence of clinically important phenomena like apoptosis and the large offspring syndrome. This review attempts to focus on the morphogenetic embryo development and gene expression profile in the in vitro-produced bovine embryos, with special emphasis on the different parameters that may alter gene expression pattern during the critical period of in vitro culture. The effect of the in vitro system, as reflected by some clinically important phenomena like apoptosis, is also discussed.

187 INCREASED NUMBER OF EXPANDED BOVINE BLASTOCYSTS IN SOF AND CR1 RELATIVE TO KSOM

2007 ◽  
Vol 19 (1) ◽  
pp. 210
Author(s):  
D. M. Kohl ◽  
R. L. Monson ◽  
L. E. Enwall ◽  
J. J. Rutledge
Keyword(s):  
Gene Expression ◽  
Culture Media ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Culture Conditions ◽  
Pregnancy Rates ◽  
Embryo Morphology ◽  
Bovine Embryos ◽  
Chi Square Analysis

Assessment of morphological stage grade is a subjective procedure. Stage grade is of vital importance to, among other things, recipient synchrony for the purpose of establishing successful pregnancies. Asynchronous embryo transfer has led to decreases in pregnancy rates (Farin et al. 1995 Biol. Reprod. 52, 676–682) and has been implicated in contributing to large offspring syndrome (Young et al. 1996 Theriogenology 45, 231). Differences in embryo kinetics based on culture conditions have been well documented (Mello et al. 2005 Reprod. Fert. Dev. 17, 221 abst). Whether such differences are the result of species, breed, metabolic stress, sire effects, or separation from an in vivo environment has yet to be determined. The correlation between oxygen respiration rates and embryo morphology as well as embryo diameter in bovine embryos produced in vitro has shown promise in the development of a more objective predictor of embryo quality and perhaps pregnancy initiation (Lopes et al. 2005 Reprod. Fert. Dev. 17, 151 abst). As well, recent examination of gene expression patterns of in vitro-derived bovine embryos seems to indicate that longer periods of in vitro culture are associated with lower rates of embryo survival (Lonergan et al. 2006 Theriogenology 65, 137–152). We hypothesize that differences do exist in the number, rate, and morphological appearance of blastocysts and that these parameters are in large part based on culture conditions in vitro. The objective of this experiment was to determine the timing and distribution of blastocyst formation of in vitro-produced bovine embryos cultured in SOF8, CR18AA, and KSOM8, under a standard incubation environment. Bovine ovaries from a local abattoir were aspirated and matured for 18-22. Oocytes were fertilized with frozen-thawed Percoll-separated semen from a Holstein bull. Presumptive zygotes were vortexed to remove cumulus cells and placed into 3 different culture media in a highly humidified atmosphere containing 20% oxygen, 5% carbon dioxide, and compressed air at 38.5�C. Embryos were evaluated specifically at 168 h post-insemination (Day 7) and assigned a morphological stage grade (IETS) to determine fixed time point differences. A total of 6 complete replicates were performed. Only embryos exhibiting the presence of a blastocoel at this time were documented (early blast, mid-blast, expanded blast). At 168 h post-insemination, there were no significant differences in the total number of embryos reaching early or mid-blast stage in any of the media. However, chi-square analysis revealed an increase in the number of expanded blastocysts in SOF (n = 813) and CR1 (n = 838) treatments compared to KSOM (n = 824; P &lt; 0.0001). Expanded blastocysts in SOF were also greater in number than in CR1 (P &lt; 0.05). Embryo selection based on development to the expanded blastocyst stage on Day 7 may prove useful in increasing pregnancy rates, and may validate qualitative correlations based on oxygen consumption and gene expression profiles for embryos produced in vitro.

24 GLOBAL TRANSCRIPT PROFILING OF CLONED BOVINE BLASTOCYSTS USING AFFYMETRIX GENECHIP TECHNOLOGY

2006 ◽  
Vol 18 (2) ◽  
pp. 120
Author(s):  
Z. Beyhan ◽  
P. Ross ◽  
A. Iager ◽  
A. Kocabas ◽  
K. Cunniff ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Expression Profiles ◽  
Cell Structure ◽  
Donor Cell ◽  
Lipid Binding ◽  
Differentially Expressed ◽  
Gtp Binding ◽  
Cloned Bovine

Identification of genes implicated in the biological processes of somatic cell nuclear transfer will improve our understanding of reprogramming events, i.e. the transformation of a lineage-committed cell into a pluripotent one. In addition, the gene expression profile of cloned embryos can help explain the widely reported developmental failures in cloned animals. In this study, we investigated global gene expression profiles of bovine in vitro-fertilized and cloned embryos using Gene Chip Bovine Genome Arrays (Affymetrix, Inc., Santa Clara, CA, USA). For the generation of cloned bovine blastocysts from two adult fibroblast lines (C and D), we employed methods previously proven to generate live offspring and compared these offspring to in vitro-produced blastocysts. Total RNA isolated from groups of 10 blastocysts was amplified by a template-switching PCR. Amplified cDNAs were used to synthesize biotin-labeled antisense RNAs (aRNAs) during and in vitro transcription reaction. Labeled aRNAs were hybridized to microarrays as described by the manufacturer. Experiments were performed in four replicates. Expression data were analyzed using the Significance Analysis of Microarrays (SAM; Tusher et al. 2001 Proc. Natl. Acad. Sci. 98, 5116-5121) procedure and software. Overall, 48.4% and 46% of 23 000 bovine transcripts spotted on the arrays were present in cloned and in in vitro-produced control blastocysts, respectively. The SAM procedure identified 43 genes that changed at least 1.5-fold, with an estimated false discovery rate (FDR) of 20%. Comparison of gene expression between NT embryos produced from two different cell lines and IVF controls with the same criteria revealed 6 (clones from cell line C vs. IVF) and 46 (clones from cell line D vs. IVF) differentially expressed genes. The number of transcripts expressed differentially between the cloned embryos with different donor cell origin was 437. Of the 43 differentially expressed transcripts in cloned blastocysts, 13 have unknown functions and the rest of the genes related to cell structure (tuftelin, desmoplakin), cell cycle/mitosis (Kinesin like 4, katanin, stathmin, PCNA), energy metabolism (lactate dehydrogenase, ATPsynthase, lipid-binding protein, keto acid dehydrogenase E1, metallothionein), and cell signaling (GTP-binding protein1, GTP binding stimulatory protein). Our results indicate that expression profiles of cloned blastocysts could be affected by somatic donor cell.

171 THE EFFECT OF TRICHOSTATIN A ON THE DEVELOPMENT AND THE GLOBAL GENE EXPRESSION PATTERNS IN MOUSE EMBRYOS DEVELOPING IN VITRO

2008 ◽  
Vol 20 (1) ◽  
pp. 165
Author(s):  
X. S. Cui ◽  
X. Y. Li ◽  
T. Kim ◽  
N.-H. Kim
Keyword(s):  
Gene Expression ◽  
Trichostatin A ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Gene Expression Profiles ◽  
Mouse Embryos ◽  
Differentially Expressed ◽  
Gene Expression Patterns ◽  
Cell Stage

Trichostatin A (TSA) is an inhibitor of histone deacetylase and is able to alter gene expression patterns by interfering with the removal of acetyl groups from histones. The aim of this study was to determine the effect of TSA treatment on the development and gene expression patterns of mouse zygotes developing in vitro. The addition of 100 nm TSA to the culture medium did not affect the cleavage of mouse embryos (TSA treatment, 148/150 (99%) v. control, 107/107 (100%)); however, embryos that were treated with TSA arrested at the 2-cell stage (145/148, 98%). We estimated the number of nuclei in control and TSA-treated embryos by propidium iodide staining, taking into account the presence of any cells with two or more nuclei. At 62–63 h post-hCG stimulation, control zygotes had developed to the 4-cell stage and exhibited one nucleus in each blastomere, indicative of normal development. In contrast, we observed tetraploid nuclei in at least one blastomere in 20.8% (11/53) of the embryos that had been treated with TSA. At 28–29 h post-hCG stimulation (metaphase of the 1-cell stage), there was no difference in the mitotic index (as determined by analyzing the microtubule configuration) in the TSA group compared to the control group. At the 2-cell stage, however, we did not observe mitotic spindles and metaphase chromatin in embryos in the TSA treatment group compared to the controls. Interestingly, when embryos were cultured in TSA-free medium from 35 h post-hCG stimulation (S- or early G2-phase of the 2-cell stage) onward, almost all of them (47/50) developed to the blastocyst stage. In contrast, when embryos were cultured in TSA-free medium from 42 h post-hCG stimulation (middle G2-phase of the 2-cell stage) onward, they did not develop to the 4-cell stage. We used Illumina microarray technology to analyze the gene expression profiles in control and TSA-treated late 2-cell-stage embryos. Applied Biosystems Expression System software was used to extract assay signals and assay signal-to-noise ratio values from the microarray images. Our data showed that 897 genes were significantly (P < 0.05; 2-sample t-test) up- or down-regulated by TSA treatment compared to controls. Analysis using the PANTHER classification system (https://panther.appliedbiosystems.com) revealed that the 575 genes that were differentially expressed in the TSA group compared to the control were classified as being associated with putative biological processes or molecular function. Overall, in terms of putative biological processes, more nucleoside, nucleotide, and nucleic acid metabolism, protein metabolism and modification, signal transduction, developmental process, and cell cycle genes were differentially expressed between the TSA and control groups. In terms of putative molecular function, more nucleic acid-binding transcription factor and transferase genes were differentially expressed between the groups. The results collectively suggest that inhibition of histone acetylation in mouse embryos affects gene expression profiles at the time of zygotic genome activation, and this subsequently affects further development.

3 IN VITRO MATURATION ALTERS GENE EXPRESSION IN MOUSE OOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 82
Author(s):  
M. Paczkowski ◽  
C. Bidwell ◽  
D. Spurlock ◽  
J. Waddell ◽  
R. L. Krisher
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Dna Methyltransferase ◽  
In Vitro Maturation ◽  
Fold Increase ◽  
Developmental Competence ◽  
Differentially Expressed ◽  
Dna Methyltransferase 1

The in vitro culture environment significantly impacts nuclear maturation, fertilization, embryonic development, and epigenetic competence; however, our knowledge of the effects of in vitro maturation on oocyte developmental competence, and specifically cytoplasmic maturation, is limited. The objective of this experiment was to identify alterations in the transcriptome of oocytes matured in vitro compared to those matured in vivo that correlate to developmental competence. Immature oocytes were collected from Day 26 and 7-8-week-old B6D2F1 mice 48 h post-pregnant mare serum gonadotropin (PMSG) administration and matured for 16 h in Gmat supplemented with 0.5 mm citric acid, 0.5 mm cysteamine, 100 ng mL–1 epidermal growth factor (EGF), 0.05% insulin-transferrin-selenium (ITS; v/v), 0.01% recombumin (v/v) and 2 mg mL–1 fetuin. In vivo-matured oocytes from females of the same ages were collected from the oviducts 62 h post-PMSG and 14 h post-hCG and mating to vasectomized males. In vivo- and in vitro-matured oocytes were identified visually by the presence of the first polar body. Mature oocytes were pooled into three groups of 150 oocytes per treatment and lysed; poly A+ RNA was extracted. Samples were processed through two cycles of linear amplification and hybridized to the GeneChip� Mouse Genome 430 2.0 Array (Affymetrix, Inc., Santa Clara, CA, USA), with three arrays per treatment. Microarray data were sorted and filtered to include genes that were classified as having two present calls per treatment. The data were then normalized to the chip median and analyzed using a one-way analysis of variance; the level of significance was calculated at P < 0.01. In total, 2.17% (482/22170) and 1.61% (358/22170) of genes were differentially expressed between in vitro- and in vivo-matured oocytes in Day 26 and 7–8-week-old mice, respectively. However, 72.82% (351/482) and 67.87% (243/358) of differentially expressed genes had increased abundance in the in vitro- and in vivo-matured oocytes, respectively. Transcripts involved in gene expression, cellular growth and proliferation, and cellular development were increased in in vivo-matured oocytes from both age groups compared to those matured in vitro. Cell death was one of the higher ranking functional groups increased in the 7–8-week-old in vitro-matured oocytes compared to the 7–8-week-old in vivo-matured oocytes. Specific genes altered by in vitro maturation conditions in Day 26 oocytes were DNA methyltransferase 1 (>7-fold increase in vivo), caspase 8 (>4-fold increase in vivo), and eukaryotic translation initiation factor 1B (>4-fold increase in vivo). DNA methyltransferase 1 and ubiquitin-conjugating enzyme E2T were significantly increased in in vivo-matured 7–8-week-old oocytes (>3-fold and >5-fold, respectively). These results indicate that gene expression is altered in oocytes matured in vitro compared to those matured in vivo. Based on the functional annotations of genes differentially expressed, dysregulation of gene expression in the oocyte resulting in altered DNA methylation and an up-regulation in cell death pathways are potential developmental mechanisms influenced by in vitro culture conditions that correlate to reduced embryonic developmental potential.

152 SPECIES-SPECIFIC DIFFERENCES IN THE METHYLATION REPROGRAMMING DURING EARLY PRE-IMPLANTATION DEVELOPMENT

2017 ◽  
Vol 29 (1) ◽  
pp. 184
Author(s):  
S. Canovas ◽  
E. Ivanova ◽  
S. Garcia-Martinez ◽  
R. Romar ◽  
N. Fonseca-Balvis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Expression Profiles ◽  
Differential Expression Analysis ◽  
Gene Expression Pattern ◽  
Reproductive Technologies ◽  
Global Methylation ◽  
Species Specific

Studies in mouse and human have shown extensive DNA methylation reprogramming in pre-implantation development followed by remethylation from implantation. However, the extent to which such reprogramming is conserved in mammals and the timing of demethylation and remethylation are unknown. As part of a major objective to characterise methylation dynamics in the bovine and porcine species from the oocyte to the blastocyst stage, we aimed here to compare the distribution of methylation at single-base resolution in both species at Day 7.5 of development. The DNA methylation profiles were obtained from individual blastocysts at Day 7.5 [pig: 3 in vivo, 3 in vitro; cow: 3 in vivo, 3 in vitro, 3 inner cell mass (ICM) and 3 trophoectoderm (TE) dissected from in vitro blastocysts] using the post-bisulphite adaptor tagging method and Illumina sequencing. For oocytes, data (GEO: GSE63330) from Schroeder et al. 2015 were analysed. Raw sequences were mapped, methylation calls made using Bismark and data analysis and visualisation was done within the SeqMonk platform. Gene expression profiles from individual blastocysts (3 pig, 3 cow) were obtained by RNA-seq. Annotated mRNA features were quantitated in SeqMonk and these were fed into DESeq2 for differential expression analysis (P < 0.05) as previously reported (Love et al. 2014 Genome Biol. 15, 550). Global methylation levels in whole blastocysts differed substantially between porcine and bovine embryos (in vivo: 12.33 ± 3.6 v. 28.33 ± 3.5%; in vitro: 15.02 ± 3.3 v. 24.41 ± 4.1%). In addition, the distribution of methylation differed: the pattern of cytosine methylated seemed random in the porcine genome, but was highly structured in the bovine genome, with methylation predominantly over gene bodies, resembling the profile previously reported in oocytes (Schroeder et al. 2015 PLoS Genet. 11, e1005442). Regarding correlation analysis, gene expression versus methylation were plotted. It suggested that gene body methylation reflected gene expression pattern in oocytes as well as in bovine blastocysts. Pair-wise comparison of isolated ICM and TE was filtered to require 5% change, and replicate set statistics were applied. This revealed very similar total and regional methylation levels in the 2 compartments, indicating that remethylation does not initiate preferentially in one compartment in bovine pre-implantation embryos. This confirms, from a viewpoint of the genome-wide DNA methylation, what has been observed in mouse for specific genes: the trophoblast-specific DNA methylation occurs after the segregation of the TE and ICM (Nakanishi et al. 2012 Epigenetics 7, 173–183). Our study is the first to provide whole genome methylation profiles from single blastocysts of economically important livestock species. Our data demonstrate that methylation reprogramming in early pre-implantation development is species specific. Knowledge of these specific patterns may have high importance when decisions are taken regarding the use of assisted reproductive technologies, cloning, or generation of transgenic animals. This work was funded by AGL2015–66341-R (MINECO-FEDER), PRX14/00348 (MECD), 19595/EE/14 (F. Séneca).

Patients who failed to conceive following an in vitro fertilization cycle can be clustered into different failure causes using gene expression hierarchical analysis†

2020 ◽  
Vol 103 (3) ◽  
pp. 599-607
Author(s):  
Chloé S Fortin ◽  
Scot Hamilton ◽  
Martin Laforest ◽  
Marie-Claude Léveillé ◽  
Marc-André Sirard
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Clustering Analysis ◽  
Gene Expression Pattern ◽  
Hierarchical Analysis ◽  
Stimulation Protocol ◽  
Follicular Cells ◽  
Vitro Fertilization ◽  
Different Response

Abstract The patient’s response to an IVF stimulation protocol is highly variable and thus difficult to predict. When a cycle fails, there are often no apparent or obvious reasons to explain the failure. Having clues on what went wrong during stimulation could serve as a basis to improve and personalize the next protocol. This exploratory study aimed to investigate if it is possible to distinguish different failure causes or different follicular responses in a population of nonpregnant IVF patients. Using qRT-PCR, we analyzed a panel of genes indicative of different failure causes in patients who did not achieve pregnancy following an IVF cycle. For each patient, a pool of follicular cells from all aspirated follicles was used as a sample which gives a global picture of the patient’s ovary and not a specific picture of each follicle. We performed hierarchical clustering analysis to split the patients according to the gene expression pattern. Hierarchical analysis showed that the population of nonpregnant IVF patients could be divided into three clusters. Gene expression was significantly different, and each cluster displayed a particular gene expression pattern. Follicular cells from patients in clusters 1, 2 and 3 displayed respectively a pattern of gene expression related to large incompetent follicles with a higher apoptosis (over matured), to follicles not ready to ovulate (under mature) and to an excess of inflammation with no visible symptoms. This study reinforces the idea that women often have different response to the same protocol and would benefit from more personalized treatments.

Benign Ethnic Neutropenia in Individuals of African Descent: Incidence, Granulocyte Mobilization, and Gene Expression Profiling.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3069-3069 ◽  
Author(s):  
Matthew Hsieh ◽  
Kay Chin ◽  
Beth Link ◽  
David Stroncek ◽  
Ena Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ethnic Groups ◽  
Expression Pattern ◽  
Neutrophil Count ◽  
Expression Profiles ◽  
African Descent ◽  
Gene Expression Pattern ◽  
Protein Array ◽  
Nhanes Iii ◽  
Normal Volunteers

Abstract Introduction: Benign ethnic neutropenia (BEN) is a condition observed in individuals of African descent, and is characterized by a reduced absolute neutrophil count (ANC) less than 1500/uL in the absence of secondary causes. In contrast to other causes of neutropenia, BEN does not increase risk of oral or systemic infections. BEN has been described in up to 40% of certain ethnic groups, but its incidence in African Americans is unknown. Additionally, the pathogenesis is currently unknown. Through this study, we wish to determine the incidence of BEN, compare the neutrophil increment after dexamethasone (Dex) and granulocyte-colony stimulating factor (G-CSF), and compare peripheral blood (PB) granulocyte gene expression profiles by microarray and PB protein array to controls in order to better understand BEN. Methods: Complete blood count (CBC) data were obtained from the National Health and Nutrition Examination Survey (NHANES) III. White blood cell and absolute neutrophil counts were compared in non-Hispanic whites, non-Hispanic blacks, Mexican-Americans, and others. Normal volunteers of African descent were recruited through an IRB approved study at NIH. Each volunteer underwent three PB leukaphereses: baseline, one day after 8mg of Dex, and one day after 5ug/kg G-CSF. CBCs were measured before and after each drug treatment. Granulocytes were enriched by gravity sedimentation with 6% hetastarch, hypotonic saline lysis of erythrocytes, and density gradient centrifugation using Ficoll-Pague. Granulocyte mRNA were applied to Affymetrix U133 plus 2.0 chips following extraction using a phenol-chloroform based method (RNA Stat 60, Tel-Test) and DNA removal. The gene expression pattern of BEN was compared to that of normals (ANC &gt;4000/uL). Results: The NHANES III sample size included 25,925 individuals. Mean ANC for the each ethnic groups were 4.55 (white), 3.67 (black), 4.80 (Mex-Am), and 4.52 (others) k/uL. The incidence of neutropenia (&lt;1500/uL) was 0.25% (white), 4.05% (black), 0.35% (Mex-Am), and 0.98% (others). Our local incidence of BEN was 5.7% (4 of 70 volunteers screened). The mean WBC in our BEN were 4.13 +/− 0.72 (baseline), 8.57 +/− 0.59 (Dex), and 16.9 +/− 2.3 (G-CSF) k/uL, compared to those of normals, 7.56 +/− 0.95 (baseline), 12.17 +/− 2.13 (Dex), and 29.22 +/− 4.8 (G-CSF) k/uL. The absolute neutrophil increment of BEN volunteers was 4.95 k/uL (94.7% of neutrophil increment in normal volunteers) after Dex, and 12.7 k/uL (60.2% of normal) after G-CSF. Gene expression profile comparisons showed that at baseline, several genes were upregulated more than 10 fold in BEN, including apoptotic factors, metalloproteases 8 and 13, TREML 3&4, TGF-beta, and endothelial factors (endothelin 1, endothelin receptor B, and fibroblast growth factors). These differences were less pronounced after Dex or G-CSF. Additionally, gene expression pattern is markedly different after Dex vs. G-CSF, regardless of the neutrophil count. Conclusions: Our results establish the incidence of BEN in the US at approximately 4%, significantly lower than that suggested by prior small reports. Additionally, the neutrophil increment is much lower in BEN following stimulation with G-CSF, suggesting the mechanism of granulocyte mobilization differs between Dex and G-CSF. Neutrophil mRNA in BEN showed increased expression of apoptotic, endothelial, and some leukocyte specific transcripts. Real-time PCR of candidate genes, accrual of more BEN subjects, and analyses of protein array are ongoing.

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