A novel culture medium with reduced nutrient concentrations supports the development and viability of mouse embryos

Further refinement of culture media is needed to improve the quality of embryos generated in vitro. Previous results from our laboratory demonstrated that uptake of nutrients by the embryo is significantly less than what is supplied in traditional culture media. Our objective was to determine the impact of reduced nutrient concentrations in culture medium on mouse embryo development, metabolism, and quality as a possible platform for next generation medium formulation. Concentrations of carbohydrates, amino acids, and vitamins could be reduced by 50% with no detrimental effects, but blastocyst development was impaired at 25% of standard nutrient provision (reduced nutrient medium; RN). Addition of pyruvate and L-lactate (+PL) to RN at 50% of standard concentrations restored blastocyst development, hatching, and cell number. In addition, blastocysts produced in RN\u2009+PL contained more ICM cells and ATP than blastocysts cultured in our control (100% nutrient) medium; however, metabolic activity was altered. Similarly, embryos produced in the RN medium with elevated (50% control) concentrations of pyruvate and lactate in the first step medium and EAA and Glu in the second step medium were competent to implant and develop into fetuses at a similar rate as embryos produced in the control medium. This novel approach to culture medium formulation could help define the optimal nutrient requirements of embryos in culture and provide a means of shifting metabolic activity towards the utilization of specific metabolic pathways that may be beneficial for embryo viability

Risk profiles and one-year outcomes of patients with newly diagnosed atrial fibrillation in India: Insights from the GARFIELD-AF Registry.

BACKGROUND: The Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF) is an ongoing prospective noninterventional registry, which is providing important information on the baseline characteristics, treatment patterns, and 1-year outcomes in patients with newly diagnosed non-valvular atrial fibrillation (NVAF). This report describes data from Indian patients recruited in this registry. METHODS AND RESULTS: A total of 52,014 patients with newly diagnosed AF were enrolled globally; of these, 1388 patients were recruited from 26 sites within India (2012-2016). In India, the mean age was 65.8 years at diagnosis of NVAF. Hypertension was the most prevalent risk factor for AF, present in 68.5% of patients from India and in 76.3% of patients globally (P < 0.001). Diabetes and coronary artery disease (CAD) were prevalent in 36.2% and 28.1% of patients as compared with global prevalence of 22.2% and 21.6%, respectively (P < 0.001 for both). Antiplatelet therapy was the most common antithrombotic treatment in India. With increasing stroke risk, however, patients were more likely to receive oral anticoagulant therapy [mainly vitamin K antagonist (VKA)], but average international normalized ratio (INR) was lower among Indian patients [median INR value 1.6 (interquartile range {IQR}: 1.3-2.3) versus 2.3 (IQR 1.8-2.8) (P < 0.001)]. Compared with other countries, patients from India had markedly higher rates of all-cause mortality [7.68 per 100 person-years (95% confidence interval 6.32-9.35) vs 4.34 (4.16-4.53), P < 0.0001], while rates of stroke/systemic embolism and major bleeding were lower after 1 year of follow-up. CONCLUSION: Compared to previously published registries from India, the GARFIELD-AF registry describes clinical profiles and outcomes in Indian patients with AF of a different etiology. The registry data show that compared to the rest of the world, Indian AF patients are younger in age and have more diabetes and CAD. Patients with a higher stroke risk are more likely to receive anticoagulation therapy with VKA but are underdosed compared with the global average in the GARFIELD-AF. CLINICAL TRIAL REGISTRATION-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01090362

73 Fatty Acid Supplementation in Culture Medium with Reduced Nutrient Concentrations Improves Bovine Blastocyst Development Compared with Standard Culture Medium

Lipids are a potent source of cellular energy and are metabolized within mitochondria via fatty acid \u3b2-oxidation, a process that also requires carnitine. Embryos metabolize lipids during pre-implantation development, but relatively little is known about the effect of fatty acid supplementation for early bovine embryogenesis in culture. The objective of this study was to evaluate the effect of lipid supplementation (via albumin) and L-carnitine (C; 5\u2009mM) during embryo culture in a novel medium with reduced concentrations of nutrients, compared with our standard culture medium (control). Following in vitro maturation and IVF, zygotes were cultured using a serum-free sequential media system (0-72\u2009h step 1; 72-168\u2009h step 2). Concentrations of salts, bicarbonate, and protein [2.5\u2009mg\u2009mL 121 fatty acid-free (FAF) or fraction V (FrV) BSA] were the same in all treatments to maintain consistent osmolarity and pH. Nutrients (glucose/fructose, citrate, lactate, pyruvate, amino acids, vitamins, and EDTA) were diluted to 6.25% of control. In addition to the control medium (100%+FAF; n\u2009=\u2009587), experimental treatments included 6.25%+FAF+C (essentially lipid free; n\u2009=\u2009573) and 6.25%+FrV+C (lipid rich; n\u2009=\u2009585). Following in vitro culture (7 reps), hatching blastocysts were stained to determine inner cell mass (ICM; SOX2+) and trophectoderm (TE; CDX2+) cell numbers. Lipid content of single expanded blastocysts was determined using gas chromatography coupled to an ISQ-LT MS/MS (GC-MS). Data (mean\u2009\ub1\u2009SEM) were analysed by ANOVA. Embryo cleavage did not differ between treatments. Blastocyst development (per cleaved embryo) was higher (P\u2009&lt;\u20090.05) after culture in lipid rich (38.3\u2009\ub1\u20091.5%) compared with control (29.6\u2009\ub1\u20092.2%) and lipid free (28.1\u2009\ub1\u20093.6%). Blastocyst hatching was reduced (P\u2009&lt;\u20090.05) in lipid free (1.4\u2009\ub1\u20090.7%) but not in lipid rich (5.2\u2009\ub1\u20091.7) compared with control (9.8\u2009\ub1\u20092.1). However, blastocysts developed in lipid rich and lipid free had reduced cell numbers compared with control: TE, 98.7\u2009\ub1\u20095.9 and 98.8\u2009\ub1\u20099.1\u2009v. 160.3\u2009\ub1\u20099.0; ICM, 19.2\u2009\ub1\u20092.9 and 25.2\u2009\ub1\u20096.1\u2009v. 43.3\u2009\ub1\u20094.0; and total cell number, 117.9\u2009\ub1\u20097.3 and 124.0\u2009\ub1\u20098.7\u2009v. 203.6\u2009\ub1\u200910.2, respectively. Analysis by GC-MS identified 40 annotated lipids (i.e. triacylglycerols and phosphatidyl cholines) that were significantly reduced in blastocysts cultured in lipid rich compared with control. In summary, blastocyst development was significantly improved after supplementation of fatty acids and L-carnitine to a medium with reduced nutrient concentrations. The mechanism underlying this phenomenon may be related to increased lipid metabolism in the low nutrient environment. Although more embryos developed in this novel medium, these blastocysts had reduced cell numbers even though blastocyst expansion and hatching were not affected. This reduced nutrient medium may provide an experimental model in which to independently study pathways controlling cell proliferation and blastocyst development. Future studies will investigate whether embryo cell number can be rescued while maintaining improved blastocyst development