Gene Expression In Human Embryos Research Articles

A shared pattern of altered gene expression in human embryos affected by mitochondrial diseases.

Does mitochondrial deficiency affect human embryonic preimplantation development? The presence of a pathogenic mitochondrial variant triggers changes in the gene expression of preimplantation human embryos, compromising their development, cell differentiation, and survival. Quantitative and qualitative anomalies of mitochondrial DNA (mtDNA) are reportedly associated with impaired human embryonic development, but the underlying mechanisms remain unexplained. Taking advantage of the preimplantation genetic testing for mitochondrial disorders in at-risk couples, we have compared gene expression of 9 human embryos carrying pathogenic variants in either mtDNA genes or nuclear genes encoding mitochondrial protein to 33 age-matched control embryos. Single-embryo transcriptomic analysis was performed on whole human blastocyst embryos donated to research. Specific pathogenic mitochondrial variants downregulate gene expression in preimplantation human embryos [566 genes in oxidative phosphorylation (OXPHOS)-deficient embryos], impacting transcriptional regulators, differentiation factors, and nuclear genes encoding mitochondrial proteins. These changes in gene expression primarily alter OXPHOS and cell survival pathways. The number of OXPHOS-deficient embryos available for the study was limited owing to the rarity of this material. However, the molecular signature shared by all these embryos supports the relevance of the findings. While identification of reliable markers of normal embryonic development is urgently needed in ART, our study prompts us to consider under-expression of the targeted genes reported here, as predictive biomarkers of mitochondrial dysfunction during preimplantation development. This work was supported by the 'Association Française contre les Myopathies (AFM-Téléthon)' and the 'La Fondation Maladies Rares'. No competing interests to declare. N/A.

Alterations of miR-16, miR-let-7a and their target genes expression in human blastocysts following vitrification and re-vitrification

Embryo cryopreservation is a widely used technique in infertility management and today is an essential part of assisted reproductive technology (ART). In some cases, re-vitrification can be applied to good quality supernumerary warmed embryos that have not been transferred in the present cycle. However, there is no study about re-vitrification impact on microRNA and gene expression in human embryos. The purpose of this study is to evaluate miR-16, miR-let7a and target genes expression in in vitro produced human blastocysts following re-vitrification.Day3 embryos obtained from ICSI cycles of fertile couples referring for family balancing program were biopsied and cultured individually. On the fourth day (post-ICSI) male ones (choices of their parents) were transferred and the females (good quality embryos) were donated for research. Donated embryos were cultured to blastocyst stage and assigned to three groups: fresh, vitrified and re-vitrification. Embryos were vitrified on Cryotech carriers. Then blastocysts of three groups were individually assessed for expression of miR-16, miR-let7a and target genes.The results showed that re-vitrification of human blastocysts did not affect the ability to re-expand in culture. In addition, significant decrease was observed in miR-16 and miR-let7a expression in re-vitrified group compared to fresh (p < 0.05). A significant upregulation of the target genes ITGβ3 and BCL-2 in re-vitrified and vitrified embryos was observed compared to the fresh group (p < 0.05). The expression of BAX as a pro-apoptotic gene showed a significant decrease in re-vitrification group comparing with the fresh one (P < 0.05).The results of this research indicated that re-vitrification of embryos changes the expression of miR-16, miR-let-7a and their target genes. These alterations include increased expression of BCl-2 and ITGβ3 genes which play important roles in embryo survival and implantation, respectively. Clinical proof of these effects requires further research.

Comprehensive chromosome screening and gene expression analysis from the same biopsy in human preimplantation embryos.

STUDY QUESTIONCan simultaneous comprehensive chromosome screening (CCS) and gene expression analysis be performed on the same biopsy of preimplantation human embryos?SUMMARY ANSWERFor the first time, CCS and reliable gene expression analysis have been performed on the same human preimplantation embryo biopsy.WHAT IS KNOWN ALREADYA single trophectoderm (TE) biopsy is routinely used for many IVF programs offering CCS for selection of only chromosomally normal embryos for transfer. Although the gene expression profiling of human preimplantation embryos has been described, to date no protocol allows for simultaneous CCS and gene expression profiling from a single TE biopsy.STUDY DESIGN, SIZE AND DURATIONThis is a proof of concept and validation study structured in two phases. In Phase 1, cell lines were subjected to a novel protocol for combined CCS and gene expression analysis so as to validate the accuracy and reliability of the proposed protocol. In Phase 2, 20 donated human blastocysts were biopsied and processed with the proposed protocol in order to obtain an accurate CCS result and characterize their gene expression profiles using the same starting material.PARTICIPANTS/MATERIALS, SETTING AND METHODA novel protocol coupling quantitative real-time PCR-based CCS and gene expression analysis using RT-PCR was designed for this study. Phase 1: six-cell aliquots of well-characterized fibroblast cell lines (GM00323, 46,XY and GM04435, 48,XY,+16,+21) were subjected to the proposed protocol. CCS results were compared with the known karyotypes for consistency, and gene expression levels were compared with levels of purified RNA from same cell lines for validation of reliable gene expression profiling. Phase 2: four biopsies were performed on 20 frozen human blastocysts previously diagnosed as trisomy 21 (10 embryos) and monosomy 21 (10 embryos) by CCS. All samples were processed with the proposed protocol and re-evaluated for concordance with the original CCS result. Their gene expression profiles were characterized and differential gene expression among embryos and early embryonic cell lineages was also evaluated.MAIN RESULTS AND THE ROLE OF CHANCECCS results from cell lines showed 100% consistency with their known karyotypes. ΔΔCt values of differential gene expression of four selected target genes from the cell lines GM4435 and GM0323 were comparable between six-cell aliquots and purified RNA (Collagen type I alpha-1 (COL1A1), P = 0.54; Fibroblast growth factor-5 (FGF5), P = 0.11; Laminin subunit beta-1 (LAMB1), P = 1.00 and Atlastin-1 (ATL1), P = 0.23). With respect to human blastocysts, 92% consistency was reported after comparing embryonic CCS results with previous diagnosis. A total of 30 genes from a human stem cell pluripotency panel were selected to evaluate gene expression in human embryos. Correlation coefficients of expression profiles from biopsies of the same embryo (r = 0.96 ± 0.03 (standard deviation), n = 45) were significantly higher than when biopsies from unrelated embryos were evaluated (r = 0.93 ± 0.03, n = 945) (P < 0.0001). Growth differentiation factor 3 (GDF3) was found to be significantly up-regulated in the inner cell mass (ICM), whereas Caudal type homebox protein-2 (CDX2), Laminin subunit alpha-1 (LAMA1) and DNA methyltransferase 3-beta (DNMT3B) showed down-regulation in ICM compared with TE. Trisomy 21 embryos showed significant up-regulation of markers of cell differentiation (Cadherin-5 (CDH5) and Laminin subunit gamma-1 (LAMC1)), whereas monosomy 21 blastocysts showed higher expression of genes reported to be expressed in undifferentiated cells (Gamma-Aminobutyric Acid Type-A Receptor Beta3 Subunit (GABRB3) and GDF3).LARGE SCALE DATAN/ALIMITATIONS, REASONS FOR CAUTIONGene expression profiles of chromosomally normal embryos were not assessed due to restrictive access to euploid embryos for research. Nonetheless, the profile of blastocysts with single aneuploidies was characterized and compared. Only 30 target genes were analyzed for gene expression in this study. Increasing the number of target genes will provide a more comprehensive transcriptomic signature and reveal potential pathways paramount for embryonic competence and correct development.WIDER IMPLICATIONS OF THE FINDINGSThis is the first time that CCS and gene expression analysis have been performed on the same human preimplantation embryo biopsy. Further optimization of this protocol with other CCS platforms and inclusion of more target genes will provide innumerable research and clinical applications, such as discovery of biomarkers for embryonic reproductive potential and characterization of the transcriptomic signatures of embryos, potentially allowing for further embryo selection prior to embryo transfer and therefore improving outcomes.STUDY FUNDING AND COMPETING INTERESTSThis study was funded by the Foundation for Embryonic Competence, Basking Ridge, NJ, USA. No conflicts of interests declared.

Mutations in BMP4 Cause Eye, Brain, and Digit Developmental Anomalies: Overlap between the BMP4 and Hedgehog Signaling Pathways

Developmental ocular malformations, including anophthalmia-microphthalmia (AM), are heterogeneous disorders with frequent sporadic or non-Mendelian inheritance. Recurrent interstitial deletions of 14q22-q23 have been associated with AM, sometimes with poly/syndactyly and hypopituitarism. We identify two further cases of AM (one with associated pituitary anomalies) with a 14q22-q23 deletion. Using a positional candidate gene approach, we analyzed the BMP4 (Bone Morphogenetic Protein-4) gene and identified a frameshift mutation (c.226del2, p.S76fs104X) that segregated with AM, retinal dystrophy, myopia, brain anomalies, and polydactyly in a family and a nonconservative missense mutation (c.278A-->G, p.E93G) in a highly conserved base in another family. MR imaging and tractography in the c.226del2 proband revealed a primary brain developmental disorder affecting thalamostriatal and callosal pathways, also present in the affected grandmother. Using in situ hybridization in human embryos, we demonstrate expression of BMP4 in optic vesicle, developing retina and lens, pituitary region, and digits strongly supporting BMP4 as a causative gene for AM, pituitary, and poly/syndactyly. Because BMP4 interacts with HH signaling genes in animals, we evaluated gene expression in human embryos and demonstrate cotemporal and cospatial expression of BMP4 and HH signaling genes. We also identified four cases, some of whom had retinal dystrophy, with "low-penetrant" mutations in both BMP4 and HH signaling genes: SHH (Sonic Hedgehog) or PTCH1 (Patched). We propose that BMP4 is a major gene for AM and/or retinal dystrophy and brain anomalies and may be a candidate gene for myopia and poly/syndactyly. Our finding of low-penetrant variants in BMP4 and HH signaling partners is suggestive of an interaction between the two pathways in humans.

P-227: Gene expression in the human preimplantation stage embryo

Microarray technology allows researchers to characterize gene expression in human embryos at developmental stages from fertilization to implantation. The information learned from such studies may provide valuable insight into embryonic abnormalities, sub optimal culture conditions and abnormal genomic activation. Our program has initiated a study to characterize gene expression patterns in human embryos fertilized and developed in vitro. For our initial attempt, morula and blastocyst stage embryos were analyzed for differences in gene expression that could be occurring during embryonic compaction and trophoblast differentiation.

RNA isolation for the study of gene expression in human embryos

Most information currently available on the human genome has been collected from adult tissues. As researchers continue to examine in vivo and in vitro development and its impact on infertility, there exists a need to study the genes that are expressed during the preimplantation stages. In the past, it has been difficult to collect this information due to the unavailability of sufficient numbers of human embryos for research. With the advent of new technologies such as microarray analysis, it is now possible to study the expression of many different genes from samples consisting of few cells, including preimplantation stage embryos.