23 research outputs found

    The use of a novel microfluidic culture device and predictive metabolic profiling as a means to improve murine embryo developmental competence in vitro

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    Successful embryo development in vitro is directly dependent on the provision of an optimal culture environment that supports coordinated embryonic cell division, metabolism, and genetic and epigenetic development. A number of attempts have been made over recent years to use microfluidic devices in IVF as a means to control the culture environment and so improve embryo developmental competence (quality) in vitro. In this study we have designed, engineered and tested a novel microfluidic device for the in vitro production of murine embryos from the 1 cell zygote stage to the blastocyst stage. Soft lithography was used to prepare microfluidic devices in polydimethylsiloxane (PDMS). The microfluidic device consists of a 400 nL circular chamber (radius 750 mm) where 10 embryos can be loaded, kept in static culture for the full period of culture and visualized by optical and fluorescent microscopy. A series of 2 experiments were conducted to evaluate the efficacy of our microfluidic device for mouse embryo culture. Cryopreserved, IVF-derived, mouse embryos of strain C57BL/6N, provided by MRC Harwell, UK were cultured in KSOM media. Microfluidic culture was used in conjunction with non-invasive analysis of glucose (G), pyruvate (P) and lactate (L) metabolism in spent zygote culture media as a means to improve embryo quality. In both experimental series, data from microfluidic cultures were compared to equivalent end point analyses of control embryos grown in conventional microdrop cultures under oil. Experiment 1: 2 cell embryos were thawed and cultured in groups of 8-10 in microfluidic devices (n=46) or 10µl control (n=32) drops for 3 days at 37oC under 5%CO2/5%O2/N2 balance. Embryos were removed to individual culture drops for 24h for analysis of energy substrate turnover using the method of Guerif et al. (PLOS ONE, 2013) followed by transfer to fibronectin-coated dishes for assessment of attachment and outgrowth according to the method of Hannan et al. (Endocrinology, 152 (12), p4948-4956, 2011). Blastocyst grade, hatching, attachment, outgrowth rates, and pyruvate and glucose consumption were assessed and were similar between device and control groups (P>0.05). However, lactate output was significantly reduced following device culture vs controls (4.1±0.8 vs 1.4±0.3 pmol/embryo/hr, P=<0.0001). GPL metabolism did not predict embryo attachment or outgrowth in either culture environment. Experiment 2: 1 cell zygotes were cultured individually overnight for analysis of GPL metabolism and assigned to culture groups based on pyruvate consumption, with 1 device and 1 microdrop group per tertile per culture with 10 embryos per group (total n=60 device and n=60 control). Following group culture, individual blastocyst pyruvate consumption was reduced (5.4±2.2 vs 12±1.5 pmol/embryo/hr, P=<0.0001). Pyruvate consumption tertile was unaffected by device culture. Device culture was non-toxic and did not affect embryo development. However, blastocyst pyruvate consumption and lactate output were reduced compared to controls. This may suggest microfluidic culture can be utilised to achieve a controlled, moderate metabolic phenotype, reducing variation between embryo metabolism

    On-chip mouse embryo culture: Evaluation of effects of uterine cells-conditioned media on embryo development and gene expression

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    Microfluidics has recently been proposed as a method to overcome the limitations of traditional oocyte and embryo culture methods. In this work, we report the use of a microfluidic polydimethylsiloxane device as promising alternative for in vitro embryo culture, and we have evaluated the effects of cells- conditioned media (CM) on embryo development. The microfluidic device was fabricated using traditional soft-lithographic technique. To produce CM, mouse uterine epithelial cells (Creative Bioarray, USA) were cultured in KSOM (Merck Millipore, UK) for 24 h. The CM was used to culture groups of 12, 1 cell murine embryos (B6C3F1xB6D2F1 strain, EmbryoTech, USA) into a microfluidic device. Control embryos were cultured in the device using KSOM. We compared blastocyst rates of embryos cultured in CM with those obtained using KSOM. The effect of treatment on embryo gene expression was assessed in cDNAs generated from individual stage matched, blastocysts using a custom, real time PCR array. Developmental ability of mouse embryos in the presence of CM was significantly higher (p<0.05) in comparison with control media. Blastocyst rates for the CM (n=15 devices, 180 embryos) and control media (n=15 devices, 180 embryos) groups were 68.9% and 45.1%, respectively. qPCR results showed that expression of Makorin Ring Finger Protein (MKRN, p=0.036), DNA Methyltransferase 3β (DNMT3β, p=0.012), DNA (Cytosine -5-)-Methyltransferase 3-Like (DNMT3L, p=0.043), Histone Acetyltransferase 1 (HAT1, p=0.006), Keratin 18 (KRT18, p=0.028), and Ubiquitin Like With PHD And Ring Finger Domains 1 (UHRF1, p=0.043) was significantly different between the treatment groups. Specifically, we observed in the CM group increased expression of DNMT3β and DNMT3L, which play an important role in early embryo development. Those finding revealed that the new microfluidic device supports mouse preimplantation embryo development in vitro. Uterine epithelial cells-conditioned medium has the potential to enhance blastocyst development. Further investigations are required to identify the mechanism of this effect

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

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    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

    A Simple Approach for COnsumption and RElease (CORE) Analysis of Metabolic Activity in Single Mammalian Embryos

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    Non-invasive assay of the consumption and release of metabolites by individual human embryos could allow selection at the cleavage stage of development and facilitate Single Embryo Transfer in clinical IVF but will require simple, high throughput, sensitive methods applicable to small volume samples. A rapid, simple, non-invasive method has therefore been devised using a standard fluorescence plate reader, and used to measure the consumption of pyruvate and glucose, and release of lactate by single bovine embryos at all stages of preimplantation development in culture; amino acid profiles have been determined using HPLC. Early embryos with an ‘intermediate’ level (6.14±0.27 pmol/embryo/h) of pyruvate uptake were associated with the highest rate (68.3%) of blastocyst development indicating that a mid “optimum” range of pyruvate consumption correlates with high viability in this bovine model

    Chapter 25 - Metabolomic Screening of Embryos to Enhance Successful Selection and Transfer

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    androgen aneuploidy antral follicles assessment assisted hatching assisted reproductive technology blastocyst blastocyst stage catheter chromosome cleavage stage clinical pregnancy rate cryopreservation culture media DHEA donor dose effect embryo culture embryo development embryo transfer Fertil Steril follicular follitropin alfa genetic GnRH agonist GnRH antagonist gonadotropin granulosa cells growth hormone Hum Reprod human embryo Human Reproduction ICSI implantation rates improve increased infertility injection IVF cycle levels LH surge live birth rates melatonin meta-analysis metabolic mitochondrial morphokinetic morphology mtDNA needle oestradiol OHSS oocyte oocyte collection oocyte maturation oocyte retrieval outcome ovarian hyperstimulation ovarian hyperstimulation syndrome ovarian reserve ovarian stimulation ovary ovulation patients percent pituitary pm/L polycystic polycystic ovary syndrome poor responders potential preimplantation progesterone protein RCTs receptor recombinant Reprod Biomed Online rFSH risk serum significant significantly sperm steroid studies supplementation syndrome transvaginal treatment trigger versus vitrification vitro fertilization women zona pelluci

    Probing morphological, genetic and metabolomic changes of in vitro embryo development in a microfluidic device

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    Assisted reproduction technologies for clinical and research purposes rely on a brief in vitro embryo culture which, despite decades of progress, remain suboptimal in comparison to the physiological environment. One promising tool to improve this technique is the development of bespoke microfluidic chambers. Here we present and validate a new microfluidic device in polydimethylsiloxane (PDMS) for the culture of early mouse embryos. Device material and design resulted embryo compatible and elicit minimal stress. Blastocyst formation, hatching, attachment and outgrowth formation on fibronectin-coated devices were similar to traditional microdrop methods. Total blastocyst cell number and allocation to the trophectoderm and inner cell mass lineages were unaffected. The devices were designed for culture of 10-12 embryos. Development rates, mitochondrial polarisation and metabolic turnover of key energy substrates glucose, pyruvate and lactate were consistent with groups of 10 embryos in microdrop controls. Increasing group size to 40 embryos per device was associated with increased variation in development rates and altered metabolism. Device culture did not perturb blastocyst gene expression but did elicit changes in embryo metabolome, which can be ascribed to substrate leaching from PDMS and warrant further investigation

    Effect of Linolenic acid during in vitro maturation of ovine oocytes: embryonic developmental potential and mRNA abundances of genes involved in apoptosis

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    PURPOSE: To study the effect of α-linolenic acid (ALA) on meiotic maturation, mRNA abundance of apoptosis-related (Bax and Bcl-2) molecules, and blastocyst formation in ovine oocytes. METHODS: A preliminary experiment was conducted to analyze the concentration of ALA in “small” (≤2 mm) and “large” (≥6 mm) follicles using gas chromatography/mass spectrometry analysis. The concentration of ALA in small and large follicles was determined to be in a range of 75.4 to 125.7 μM, respectively. In vitro maturation (IVM) of oocyte was then performed in presence of 0 (control), 10 (ALA-10), 50 (ALA-50), 100 (ALA-100), and 200 (ALA-200) μM of ALA. Meiotic maturation and mRNA abundance of Bax, and Bcl-2 genes was evaluated after 24 h of IVM. The embryonic cleavage and blastocyst formation following parthenogenetic activation were also determined for each group. RESULTS: The highest concentration of ALA (ALA-200) decreased the oocyte maturation rate compared with the control group. Analysis of apoptosis-related genes in oocytes after IVM revealed lesser transcript abundances for Bax gene, and higher transcript abundances for Bcl-2 gene in ALA-treated oocytes as compared with the control oocytes. In term of cleavage rate (considered as 2-cell progression), we did not observe any differences among the groups. However, ALA-100 group promoted more blastocyst formation as compared with the control group. CONCLUSION: Our results suggested that ALA treatment during IVM had a beneficial effect on developmental competence of ovine oocytes by increasing the blastocyst formation and this might be due to the altered abundance of apoptosis-regulatory genes
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