Cloned cattle derived from a novel zona-free embryo reconstruction system

As the demand for cloned embryos and offspring increases, the need arises for the development of nuclear transfer procedures that are improved in both efficiency and ease of operation. Here, we describe a novel zona-free cloning method that doubles the throughput in cloned bovine embryo production over current procedures and generates viable offspring with the same efficiency. Elements of the procedure include zona-free enucleation without a holding pipette, automated fusion of 5-10 oocyte-donor cell pairs and microdrop in vitro culture. Using this system, zona-free embryos were reconstructed from five independent primary cell lines and cultured either singularly (single-IVC) or as aggregates of three (triple-IVC). Blastocysts of transferable quality were obtained at similar rates from zona-free single-IVC, triple-IVC, and control zona-intact embryos (33%, 25%, and 29%, respectively). In a direct comparison, there was no significant difference in development to live calves at term between single-IVC, triple-IVC, and zona-intact embryos derived from the same adult fibroblast line (10%, 13%, and 15%, respectively). This zona-free cloning method could be straightforward for users of conventional cloning procedures to adopt and may prove a simple, fast, and efficient alternative for nuclear cloning of other species as well

A comparison of polarized and non-polarized human endometrial monolayer culture systems on murine embryo development

BACKGROUND: Co-culture of embryos with various somatic cells has been suggested as a promising approach to improve embryo development. Despite numerous reports regarding the beneficial effects of epithelial cells from the female genital tract on embryo development in a co-culture system, little is known about the effect of these cells when being cultured under a polarized condition on embryo growth. Our study evaluated the effects of in vitro polarized cells on pre-embryo development. METHODS: Human endometrial tissue was obtained from uterine specimens excised at total hysterectomy performed for benign indications. Epithelial cells were promptly isolated and cultured either on extra-cellular matrix gel (ECM-Gel) coated millipore filter inserts (polarized) or plastic surfaces (non-polarized). The epithelial nature of the cells cultured on plastic was confirmed through immunohistochemistry, and polarization of cells cultured on ECM-Gel was evaluated by transmission electron microscopy (TEM). One or two-cell stage embryos of a superovulated NMRI mouse were then flushed and placed in culture with either polarized or non-polarized cells and medium alone. Development rates were determined for all embryos daily and statistically compared. At the end of the cultivation period, trophectoderm (TE) and inner cell mass (ICM) of expanded blastocysts from each group were examined microscopically. RESULTS: Endometrial epithelial cells cultured on ECM-Gel had a highly polarized columnar shape as opposed to the flattened shape of the cells cultured on a plastic surface. The two-cell embryos cultured on a polarized monolayer had a higher developmental rate than those from the non-polarized cells. There was no statistically significant difference; still, the blastocysts from the polarized monolayer, in comparison with the non-polarized group, had a significantly higher mean cell number. The development of one-cell embryos in the polarized and non-polarized groups showed no statistically significant difference. CONCLUSION: Polarized cells could improve in vitro embryo development from the two-cell stage more in terms of quality (increasing blastocyst cellularity) than in terms of developmental rate

Improved in vitro development of cloned bovine embryos using S-adenosylhomocysteine, a non-toxic epigenetic modifying reagent.

In this study, fibroblast cells were stably transfected with mouse POU5F1 promoter-driven enhanced green fluorescent protein (EGFP) to investigate the effect of S-adenosylhomocysteine (SAH), the reversible non-toxic inhibitor of DNA-methyltransferases (DNMTs), at different intervals post-fusion on in vitro development of cloned bovine embryos. Treatment with SAH for 12 hr resulted in 54.6 ± 7.7% blastocyst production, which was significantly greater than in vitro fertilized embryos (IVF: 37.2 ± 2.7%), cloned embryos treated with SAH for 72 hr (31.0 ± 7.6%), and control cloned embryos (34.6 ± 3.6%). The fluorescence intensities of the EGFP-POU5F1 reporter gene at all intervals of SAH treatment, except of 72 hr, were significantly higher than control somatic cell nuclear transfers (SCNT) embryos. The intensity of DNA-methylation in cloned embryos treated with SAH for 48 hr was similar to that of IVF embryos, and was significantly lower than the other SCNT groups. The levels of H3K9 acetylation in all SCNT groups were significantly lower than IVF embryos. Real-time PCR analysis of gene expression revealed significantly higher expression of POU5F1 in cloned versus IVF blastocysts. Neither embryo production method (SCNT vs. IVF) nor the SAH treatment interval affected expression of the BCL2 gene. Cloned embryos at all intervals of SAH treatment, except for 24 hr, had significantly increased VEGF transcript compared to IVF and control SCNT embryos. It was suggested that the time interval of DNMT inhibition may have important consequences on different in vitro features of bovine SCNT, and the improving effects of DNMT inhibition on developmental competency of cloned embryos are restricted to a specific period of time preceding de novo methylation. Mol. Reprod. Dev. 78:576–584, 2011

Preserving and Using Germplasm and Dissociated Embryonic Cells for Conserving Caribbean and Pacific Coral

Coral reefs are experiencing unprecedented degradation due to human activities, and protecting specific reef habitats may not stop this decline, because the most serious threats are global (i.e., climate change), not local. However, ex situ preservation practices can provide safeguards for coral reef conservation. Specifically, modern advances in cryobiology and genome banking could secure existing species and genetic diversity until genotypes can be introduced into rehabilitated habitats. We assessed the feasibility of recovering viable sperm and embryonic cells post-thaw from two coral species, Acropora palmata and Fungia scutaria that have diffferent evolutionary histories, ecological niches and reproductive strategies. In vitro fertilization (IVF) of conspecific eggs using fresh (control) spermatozoa revealed high levels of fertilization (>90% in A. palmata; >84% in F. scutaria; P>0.05) that were unaffected by tested sperm concentrations. A solution of 10% dimethyl sulfoxide (DMSO) at cooling rates of 20 to 30°C/min most successfully cryopreserved both A. palmata and F. scutaria spermatozoa and allowed producing developing larvae in vitro. IVF success under these conditions was 65% in A. palmata and 53% in F. scutaria on particular nights; however, on subsequent nights, the same process resulted in little or no IVF success. Thus, the window for optimal freezing of high quality spermatozoa was short (∼5 h for one night each spawning cycle). Additionally, cryopreserved F. scutaria embryonic cells had∼50% post-thaw viability as measured by intact membranes. Thus, despite some differences between species, coral spermatozoa and embryonic cells are viable after low temperature (−196°C) storage, preservation and thawing. Based on these results, we have begun systematically banking coral spermatozoa and embryonic cells on a large-scale as a support approach for preserving existing bio- and genetic diversity found in reef systems

Second asymptomatic carotid surgery trial (ACST-2): a randomised comparison of carotid artery stenting versus carotid endarterectomy

Background: Among asymptomatic patients with severe carotid artery stenosis but no recent stroke or transient cerebral ischaemia, either carotid artery stenting (CAS) or carotid endarterectomy (CEA) can restore patency and reduce long-term stroke risks. However, from recent national registry data, each option causes about 1% procedural risk of disabling stroke or death. Comparison of their long-term protective effects requires large-scale randomised evidence. Methods: ACST-2 is an international multicentre randomised trial of CAS versus CEA among asymptomatic patients with severe stenosis thought to require intervention, interpreted with all other relevant trials. Patients were eligible if they had severe unilateral or bilateral carotid artery stenosis and both doctor and patient agreed that a carotid procedure should be undertaken, but they were substantially uncertain which one to choose. Patients were randomly allocated to CAS or CEA and followed up at 1 month and then annually, for a mean 5 years. Procedural events were those within 30 days of the intervention. Intention-to-treat analyses are provided. Analyses including procedural hazards use tabular methods. Analyses and meta-analyses of non-procedural strokes use Kaplan-Meier and log-rank methods. The trial is registered with the ISRCTN registry, ISRCTN21144362. Findings: Between Jan 15, 2008, and Dec 31, 2020, 3625 patients in 130 centres were randomly allocated, 1811 to CAS and 1814 to CEA, with good compliance, good medical therapy and a mean 5 years of follow-up. Overall, 1% had disabling stroke or death procedurally (15 allocated to CAS and 18 to CEA) and 2% had non-disabling procedural stroke (48 allocated to CAS and 29 to CEA). Kaplan-Meier estimates of 5-year non-procedural stroke were 2·5% in each group for fatal or disabling stroke, and 5·3% with CAS versus 4·5% with CEA for any stroke (rate ratio [RR] 1·16, 95% CI 0·86–1·57; p=0·33). Combining RRs for any non-procedural stroke in all CAS versus CEA trials, the RR was similar in symptomatic and asymptomatic patients (overall RR 1·11, 95% CI 0·91–1·32; p=0·21). Interpretation: Serious complications are similarly uncommon after competent CAS and CEA, and the long-term effects of these two carotid artery procedures on fatal or disabling stroke are comparable. Funding: UK Medical Research Council and Health Technology Assessment Programme

Optimised medical therapy alone versus optimised medical therapy plus revascularisation for asymptomatic or low-to-intermediate risk symptomatic carotid stenosis (ECST-2): 2-year interim results of a multicentre randomised trial

BackgroundCarotid revascularisation, comprising either carotid endarterectomy or stenting, is offered to patients with carotid stenosis to prevent stroke based on the results of randomised trials conducted more than 30 years ago. Since then, medical therapy for stroke prevention has improved. We aimed to assess whether patients with asymptomatic and symptomatic carotid stenosis with a low or intermediate predicted risk of stroke, who received optimised medical therapy (OMT), would benefit from additional revascularisation.MethodsThe Second European Carotid Surgery Trial (ECST-2) is a multicentre randomised trial with blinded outcome adjudication, which was conducted at 30 centres with stroke and carotid revascularisation expertise in Europe and Canada. Patients aged 18 years or older with asymptomatic or symptomatic carotid stenosis of 50% or greater, and a 5-year predicted risk of ipsilateral stroke of less than 20% (estimated using the Carotid Artery Risk [CAR] score), were recruited. Patients were randomly assigned to either OMT alone or OMT plus revascularisation (1:1) using a web-based system. The primary outcome for this 2-year, interim analysis was a hierarchical outcome composite of: (1) periprocedural death, fatal stroke, or fatal myocardial infarction; (2) non-fatal stroke; (3) non-fatal myocardial infarction; or (4) new silent cerebral infarction on imaging. Analysis was by intention-to-treat using the win ratio—ie, each patient in the OMT alone group was compared as a pair with each patient in the OMT plus revascularisation group, with a win declared for the patient with a better outcome within the pair (a tie was declared if neither patient in the pair had a better outcome). The win ratio was calculated as the number of wins in the OMT alone group divided by the number of wins in the OMT plus revascularisation group. This trial is registered with the ISRCTN Registry (ISRCTN97744893) and is ongoing.FindingsBetween March 1, 2012, and Oct 31, 2019, 429 patients were randomly assigned to OMT alone (n=215) or OMT plus revascularisation (n=214). One patient allocated to OMT alone withdrew consent within 48 h and was not considered further. The median age of patients was 72 years (IQR 65–78); 296 (69%) were male and 133 (31%) female. No benefit was recorded in favour of either treatment group with respect to the primary hierarchical outcome assessed 2 years after randomisation, with 5228 (11·4%) wins for the OMT alone group, 5173 (11·3%) wins for the OMT plus revascularisation group, and 35 395 (77·3%) ties between groups (win ratio 1·01 [95% CI 0·60–1·70]; p=0·97). For OMT alone versus OMT plus revascularisation, four versus three patients had periprocedural death, fatal stroke, or fatal myocardial infarction; 11 versus 16 had non-fatal stroke; seven versus five had non-fatal myocardial infarction; and 12 versus seven had new silent cerebral infarction on imaging. One periprocedural death occurred in the OMT plus revascularisation group, which was attributed to decompensated aortic stenosis 1 week after carotid endarterectomy.InterpretationNo evidence for a benefit of revascularisation in addition to OMT was found in the first 2 years following treatment for patients with asymptomatic or symptomatic carotid stenosis of 50% or greater with a low or intermediate predicted stroke risk (assessed by the CAR score). The results support treating patients with asymptomatic and low or intermediate risk symptomatic carotid stenosis with OMT alone until further data from the 5-year analysis of ECST-2 and other trials become available.FundingNational Institute for Health and Care Research; Stroke Association; Swiss National Science Foundation; Dutch Organisation for Knowledge and Innovation in Health, Healthcare and Well-Being; Leeds Neurology Foundation