Gene Expression Profiles Of Embryos Research Articles

O-273 A transcriptomic approach for embryo selection after preimplantation genetic diagnosis for aneuploidy

Abstract Study question Is it possible to predict an euploid chromosomal constitution from RNA-Seq data and identify a transcriptomic profile compatible with extended embryonic development? Summary answer It has been possible to obtain a karyotype comparable to PGT-A, in addition to acquiring a transcriptomic signature of embryos suggestive of improved implantation capacity What is known already Conventional assessment of embryo competence, based on morphology and morphokinetic, lacks knowledge of molecular aspects and faces controversy in predicting ploidy status. Understanding the embryonic transcriptome is crucial, as gene expression influences development and implantation. Preimplantation genetic testing (PGT) has improved pregnancy rates, but problems persist when high-quality euploid embryos do not reach term. In fact, around 50 to 60% implant, of which 10% miscarry. Comprehensive approaches, including RNA-Seq, offer potential to discover molecular markers of reproductive competence. Extended-embryo culture platforms up to day 14 can be utilized as a proxy to study embryo development at post-implantation stages Study design, size, duration Prospective pilot cohort study conducted from March 2023 to August 2023. A total of 30 vitrified human blastocysts with previous PGT-A diagnosis on day 5 (D5) or day 6 (D6) of development were analysed: n = 15 euploid and n = 15 aneuploid embryos. Finally, 21 embryo samples were included in the study (n = 10 euploid and n = 11 aneuploid), the rest (n = 9) were excluded due to poor quality after sequencing. All embryos were donated for research purposes Participants/materials, setting, methods Following warming and re-expansion, embryos underwent a second trophectoderm biopsy. The remaining embryo was cultured until day 11 to assess its development. After biopsy analysis by RNA-Seq, karyotypes were studied, as well as differential expression gene (DEG) of the comparison: unattached embryos (n = 12) vs embryos attached (n = 9) to the plate (padj<0.05 and FC > 1). In addition, we obtained a transcriptomic signature specific to embryos with a “theoretical” capacity for sustained implantation (raw p-value<0.05 and shrunk LFC>1.1) Main results and the role of chance Digital karyotype concordance was obtained with a sensitivity of 81%, a specificity of 83%, a Kappa index of 65.5% and an area under the ROC curve of 90%. At the gene level, 76 DEGs (56 upregulated and 20 downregulated) were found in the comparison of embryos unattached to the plate in extended culture versus those that were attached. To address the functional implications of these differences, significantly deregulated pathways according to GO and KEGG categories were identified. The mural trophectoderm of the analysed blastocysts showed 63 significantly deregulated terms (30 GO-Biological Process, 13 GO-Cellular Component, 12 GO-Molecular Function and 8 KEGG pathways), displaying upregulation of autophagy, apoptosis, protein kinase and ubiquitin-like protein ligase activity, and downregulation of ribosome, spliceosome, kinetochore, segregation, and chromosome condensation processes. The first described transcriptomic signature specific to embryos with theoretical enhanced implantation capacity consists of 501 genes, including: EMP2, AURKB, FOLR1, NOTCH3, LRP2, FZD5, MDH1, APOD, GPX8, COLEC12, HSPA1A, CMTM7, BEX3, which are related to implantation and embryonic development. These findings indicate that it would be possible to identify euploid embryos with a greater capacity for implantation and development, excluding those embryos that, although they could be implanted and viable, present chromosomal alterations Limitations, reasons for caution This study included a small sample size and a remarkable variability between samples. Structural chromosomal abnormalities were not included. It is not possible to diagnose mosaic embryos. Trophectoderm biopsy does not assure the chromosomal status of the whole embryo. Maximum number of days for in vitro development was 11 Wider implications of the findings Gene expression profiles of embryos with theoretical increased implantation capacity have been obtained. Clinical implementation of this technique, which assesses chromosomal content and transcriptomic profile, could be used to significantly increase clinical outcomes in assisted reproduction, allowing informed decisions to be made about future treatments and the prospects of success Trial registration number Not applicable

A genome-wide transcriptomic analysis of embryos fathered by obese males in a murine model of diet-induced obesity

Paternal obesity is known to have a negative impact on the male’s reproductive health as well as the health of his offspring. Although epigenetic mechanisms have been implicated in the non-genetic transmission of acquired traits, the effect of paternal obesity on gene expression in the preimplantation embryo has not been fully studied. To this end, we investigated whether paternal obesity is associated with gene expression changes in eight-cell stage embryos fathered by males on a high-fat diet. We used single embryo RNA-seq to compare the gene expression profile of embryos generated by males on a high fat (HFD) versus control (CD) diet. This analysis revealed significant upregulation of the Samd4b and Gata6 gene in embryos in response to a paternal HFD. Furthermore, we could show a significant increase in expression of both Gata6 and Samd4b during differentiation of stromal vascular cells into mature adipocytes. These findings suggest that paternal obesity may induce changes in the male germ cells which are associated with the gene expression changes in the resulting preimplantation embryos.

The effects of mitochondrial DNA supplementation at the time of fertilization on the gene expression profiles of porcine preimplantation embryos.

Mitochondrial DNA (mtDNA) deficient metaphase II porcine oocytes are less likely to fertilize and more likely to arrest during preimplantation development. However, they can be supplemented with autologous populations of mitochondria at the time of fertilization, which significantly increases mtDNA copy number by the 2-cell stage due to the modulation of DNA methylation at a CpG island of the gene encoding the mtDNA-specific polymerase, POLG, and promotes preimplantation development. Although mitochondrial supplementation does not increase development rates or mtDNA copy number in oocytes with normal levels of mtDNA copy number, we tested whether this approach would also impact on chromosomal gene expression patterns in these oocytes at each stage of preimplantation development. Here, we have compared the gene expression profiles of embryos produced by mitochondrial supplementation at the time of fertilization with embryos produced by in vitro fertilization (IVF) using a panel of genes associated with different stages of preimplantation development. When compared to IVF-derived embryos, 27 (34%) genes were differentially expressed in supplemented embryos but this was restricted to one or two developmental stages. However, 53 (66%) genes were comparably expressed across all six stages and by the blastocyst stage 4 (5%) genes were differentially expressed. We conclude that additional copies of mtDNA can induce changes in gene expression at various stages of preimplantation development with the first changes occurring prior to maternal-to-zygotic transition (MZT). However, these changes appear to be transitory suggesting that some genomic resetting is taking place.

Characterization of bovine embryos cultured under conditions appropriate for sustaining human naïve pluripotency.

In mammalian preimplantation development, pluripotent cells are set aside from cells that contribute to extra-embryonic tissues. Although the pluripotent cell population of mouse and human embryos can be cultured as embryonic stem cells, little is known about the pathways involved in formation of a bovine pluripotent cell population, nor how to maintain these cells in vitro. The objective of this study was to determine the transcriptomic profile related to bovine pluripotency. Therefore, in vitro derived embryos were cultured in various culture media that recently have been reported capable of maintaining the naïve pluripotent state of human embryonic cells. Gene expression profiles of embryos cultured in these media were compared using microarray analysis and quantitative RT-PCR. Compared to standard culture conditions, embryo culture in ‘naïve’ media reduced mRNA expression levels of the key pluripotency markers NANOG and POU5F1. A relatively high percentage of genes with differential expression levels were located on the X-chromosome. In addition, reduced XIST expression was detected in embryos cultured in naïve media and female embryos contained fewer cells with H3K27me3 foci, indicating a delay in X-chromosome inactivation. Whole embryos cultured in one of the media, 5iLA, could be maintained until 23 days post fertilization. Together these data indicate that ‘naïve’ conditions do not lead to altered expression of known genes involved in pluripotency. Interestingly, X-chromosome inactivation and development of bovine embryos were dependent on the culture conditions.

Study of potential health effects of electromagnetic fields of telephony and Wi‐Fi, using chicken embryo development as animal model

The objective of this study is to investigate possible biological effects of radiofrequency electromagnetic fields (RF-EMF) as used in modern wireless telecommunication in a well-controlled experimental environment using chicken embryo development as animal model. Chicken eggs were incubated under continuous experimental exposure to GSM (1.8 GHz), DECT (1.88 GHz), UMTS (2.1 GHz), and WLAN (5.6 GHz) radiation, with the appropriate modulation protocol, using a homogeneous field distribution at a field strength of approximately 3 V/m, representing the maximum field level in a normal living environment. Radiation-shielded exposure units/egg incubators were operating in parallel for exposed and control eggs in a climatized homogeneous environment, using 450 eggs per treatment in three successive rounds per treatment. Dosimetry of the exposure (field characteristics and specific absorption rate) were studied. Biological parameters studied included embryo death during incubation, hatching percentage, and various morphological and histological parameters of embryos and chicks and their organs, and gene expression profiles of embryos on day 7 and day 18 of incubation by microarray and qPCR. No conclusive evidence was found for induced embryonic mortality or malformations by exposure to the used EMFs, or for effects on the other measured parameters. Estimated differences between treatment groups were always small and the effect of treatment was not significant. In a statistical model that ignored possible interaction between rounds and exposure units, some of the many pairwise comparisons of exposed versus control had P-values lower than 0.05, but were not significant after correction for multiple testing. Bioelectromagnetics. 38:186-203, 2017. © 2017 Wiley Periodicals, Inc.

Genome-wide microarray analysis of Atlantic cod (Gadus morhua) oocyte and embryo.

BackgroundRegulation of gene expression plays a central role in embryonic development. Early stages are controlled by gametic transcripts, which are subsequently substituted with transcripts from the genome of the zygote. Transcriptomic analyses provide an efficient approach to explore the temporal gene expression profiles in embryos and to search for the developmental regulators. We report a study of early Atlantic cod development that used a genome-wide oligonucleotide microarray to examine the composition and putative roles of polyadenylated transcripts.ResultsThe analyses were carried out in unfertilized oocytes, newly fertilized oocytes and embryos at the stages of mid-blastula transition and segmentation. Numerous genes transcribed in oocytes are involved in multiple aspects of cell maintenance and protection, including metabolism, signal perception and transduction, RNA processing, cell cycle, defense against pathogens and DNA damage. Transcripts found in unfertilized oocytes also encoded a large number of proteins implicated in cell adherence, tight junction and focal adhesion, suggesting high complexity in terms of structure and cellular interactions in embryos prior to midblastula transition (MBT). Prezygotic transcripts included multiple regulators that are most likely involved in developmental processes that take place long after fertilization, such as components of ErbB, hedgehog, notch, retinoid, TGFb, VEGF and Wnt signaling pathways, as well as transcripts involved in the development of nervous system. The major event of MBT was the activation of a large group of histones and other genes that modify chromatin structure preceding massive gene expression changes. A hallmark of events observed during segmentation was the induction of multiple transcription factors, including a large group of homeobox proteins in pace with decay of a large fraction of maternal transcripts. Microarray analyses detected a suite of master developmental regulators that control differentiation and maintenance of diverse cell lineages.ConclusionsTranscriptome profiling of the early stages in Atlantic cod revealed the presence of transcripts involved in patterning and development of tissues and organs long before activation of the zygotic genome. The switch from maternal to zygotic developmental programs is associated with large-scale modification of chromosomes.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-594) contains supplementary material, which is available to authorized users.

Use of data mining to determine changes in the gene expression profiles of rat embryos following prenatal exposure to inflammatory stimulants

Numerous clinical epidemiology and laboratory studies have demonstrated a close association between inflammation or immunity and the occurrence of hypertension. Our group has previously shown that upon intraperitoneal injection of lipopolysaccharide (LPS) or zymosan (Zym) into pregnant rats, the offspring of the experimental group exhibited higher blood pressure levels compared with the control group, and these may be associated with the effects of an accumulation of gene expression changes on female rats during pregnancy. The present study used the Affymetrix GeneChip® Rat Genome 230 2.0 Array to examine gene expression profile changes in the embryos of experimental pregnant rats intraperitoneally injected with LPS or Zym vs. the untreated controls. We discovered that genes associated with DNA replication and mRNA processing were significantly upregulated by the stimuli from analysis using GenMAPP. By contrast, genes involved in the calcium regulatory pathway in cardiac myocytes and the signaling pathway in smooth muscle contraction and relaxation were the most downregulated. Results of the microarray analysis showed that immuno-inflammatory stimuli during pregnancy affected the gene expression profiles of the embryos. These changes in gene expression may affect the developmental and metabolic status of the offspring, thereby increasing their susceptibility to hypertension and obesity.

Loss of fish actinotrichia proteins and the fin-to-limb transition

The early development of teleost paired fins is strikingly similar to that of tetrapod limb buds and is controlled by similar mechanisms. One early morphological divergence between pectoral fins and limbs is in the fate of the apical ectodermal ridge (AER), the distal epidermis that rims the bud. Whereas the AER of tetrapods regresses after specification of the skeletal progenitors, the AER of teleost fishes forms a fold that elongates. Formation of the fin fold is accompanied by the synthesis of two rows of rigid, unmineralized fibrils called actinotrichia, which keep the fold straight and guide the migration of mesenchymal cells within the fold. The actinotrichia are made of elastoidin, the components of which, apart from collagen, are unknown. Here we show that two zebrafish proteins, which we name actinodin 1 and 2 (And1 and And2), are essential structural components of elastoidin. The presence of actinodin sequences in several teleost fishes and in the elephant shark (Callorhinchus milii, which occupies a basal phylogenetic position), but not in tetrapods, suggests that these genes have been lost during tetrapod species evolution. Double gene knockdown of and1 and and2 in zebrafish embryos results in the absence of actinotrichia and impaired fin folds. Gene expression profiles in embryos lacking and1 and and2 function are consistent with pectoral fin truncation and may offer a potential explanation for the polydactyly observed in early tetrapod fossils. We propose that the loss of both actinodins and actinotrichia during evolution may have led to the loss of lepidotrichia and may have contributed to the fin-to-limb transition.

Ovarian stimulation retards postimplantation development and alters global gene expression profile of blastocysts in mouse

This study compares the quality, developmental capacity and global gene expression profile of embryos generated from superovulated and natural cycles. We found that ovarian stimulation impaired quality of blastocysts (cell number: 68.9 +/- 6.9 vs. 76.8 +/- 7.9; diameter of blastocysts: 145.3 +/- 76.9 microm vs. 152.6 +/- 65.5 microm), fetus development (rate of development to term: 45.5% vs. 69.1%; weight of 18.5-dpc fetuses: 1.23 +/- 0.03 g vs. 1.34 +/- 0.03 g) and caused 92 genes differentially expressed.

Differential gene expression profiles in embryos of the lizard Podarcis sicula under in ovo exposure to cadmium

Screening for differentially expressed genes is a straightforward approach to study the molecular basis of contaminant toxicity. In this paper, the mRNA differential display technique was applied to analyze transcriptional regulation in response to cadmium exposure in the lizard embryos. Lizard eggs may be particularly susceptible to soil contamination and in ovo exposure may interfere or disrupt normal physiological function in the developing embryo, including regulation of gene expression. Fertilized eggs of the lizard Podarcis sicula were incubated in cadmium-contaminated soil at 25 °C for 20 days. Gene expression profiling showed 5 down- and 9 up-regulated genes. Four cDNAs had no homology to known gene sequences, thus suggesting that may either encode not yet identified proteins, or correspond to untranslated regions of mRNA molecules. Four fragments exhibited significant sequence similarity with genes encoding novel proteins or ESTs derived from other vertebrates. The remaining genes are mainly involved in molecular pathways associated with processes such as membrane trafficking, signal transduction, cytoskeletal organization, cell proliferation and differentiation. Cadmium also affected the expression of factors actively involved in the regulation of the transcription machinery. Down-regulated genes are mainly associated with cellular metabolism and cell-cycle regulation and apoptosis. All of these differentially expressed genes may represent candidates that function in cadmium responses. The present study leads to an increased understanding of genes and/or the biochemical pathways involved in perturbation of embryo development following cadmium exposure.

Genomic sequences encoding two types of medaka hemopexin-like protein Wap65, and their gene expression profiles in embryos

Medaka genomic BAC clones, which contained two types of medaka hemopexin-like protein gene (Wap65), mWap65-1 and mWap65-2, were screened and their genomic sequences were determined by the shotgun strategy. The exon-intron organizations were highly conserved between both mWap65s and human hemopexin genes. The 5'-flanking regions of mWap65-1 and mWap65-2 contained various putative transcription factor binding sites including elements for developmental regulation. The expression patterns of mWap65s during embryonic development were examined by quantitative real-time PCR, demonstrating that both mWap65 transcripts were observed in early embryonic stages, but their expression patterns were different. Interestingly, in situ hybridization revealed that mWap65-2 transcripts were restricted to liver, whereas mWap65-1 transcripts were detected along the edge of pectoral fin buds and the median fin fold of tail buds in embryos at stage 32. Furthermore, we generated transgenic medaka expressing GFP driven by mWap65-1 and mWap65-2 promoters and observed GFP expression patterns during ontogeny. Although localizations of GFP varied among individuals, embryos uniformly expressed GFP 1 day after injection of mWap65-1-hrGFP and mWap65-2-hrGFP constructs, suggesting that mWap65-1 and mWap65-2 promoters were activated in very early stages. The differences between mWap65-1 and mWap65-2 in their expression profiles indicate their distinct roles during ontogeny.