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

The role of proteomics in defining the human embryonic secretome

Non-invasive gamete and embryo assessment is considered an important focus in assisted reproductive technologies (ART). Currently, the selection of embryos for transfer is based on morphological indices. Though successful, the field of ART would benefit from a non-invasive quantitative method of viability determination. Omics technologies, including transcriptomics, proteomics and metabolomics, have already begun providing evidence that viable gametes and embryos possess unique molecular profiles with potential biomarkers that can be utilized for developmental and/or viability selection. Unlike the human genome that is relatively fixed and steady throughout the human body, the human proteome, estimated at over a million proteins, is more complex, diverse and dynamic. It is the proteins themselves that contribute to the physiological homeostasis in any cell or tissue. Of particular interest in ART is the secretome, those proteins that are produced within the embryo and secreted into the surrounding environment. Defining the human embryonic secretome has the potential to expand our knowledge of embryonic cellular processes, including the complex dialogue between the developing embryo and its maternal environment, and may also assist in identifying those embryos with the highest implantation potential. Advances in proteomic technologies have allowed the non-invasive profiling of the human embryonic secretome with ongoing research focused on correlation with outcome. From a clinical perspective, embryo selection based on morphological assessment and non-invasive analysis of the human embryonic secretome may improve IVF success and lead to routine single embryo transfers

Alterations in oocyte mitochondrial number and function are related to spindle defects and occur with maternal aging in mice and humans

The objective of this work was to determine the role of mitochondria in the loss of oocyte quality with maternal aging. Our results show that mitochondrial DNA (mtDNA) copy number and function are reduced in eggs from aged mice after both in vivo and in vitro maturation. Higher incidences of spindle abnormalities were observed in old eggs. However, no correlation with egg ATP content was found. In vitro matured eggs from aged mice did not have a normal cortical distribution of active mitochondria and were subject to increased oxidative stress due to higher levels of reactive oxygen species and lower expression of glutamate-cysteine ligase, catalytic subunit (Gclc). Supplementation of antioxidants during in vitro maturation of old eggs mitigated this affect, resulting in increased mtDNA copy number and mitochondrial function, a mitochondria distribution pattern similar to young eggs, and improved chromosomal alignment. Eggs from women of advanced maternal age (AMA) had lower mitochondrial function than eggs from young women, although both age groups displayed a cortical distribution pattern of active mitochondria. In contrast to the mouse, human eggs from AMA women had higher mtDNA copy number than eggs from young women following in vitro maturation. In summary, oocytes of older females are more susceptible to perturbations in mitochondrial number and function, which are associated with increased spindle abnormalities and oxidative stress during in vitro maturation. These results demonstrate that oocyte mitochondria play a critical role in age-related infertility

Sequential nutrient restriction and provision during bovine in vitro embryo culture differentially affect blastocyst development and quality with oocytes from varied sources

We have demonstrated that bovine blastocyst development was improved after culture in medium with only 6.25% of standard carbohydrate and amino acid concentrations, supplemented with fatty acids. However, these blastocysts had lower cell numbers. We hypothesised that this was due to deficiencies in embryo metabolism at the time of blastocyst formation. Thus, our objectives were to (1) determine whether using a sequential combination of nutrient concentrations could rescue blastocyst cell number; and (2) investigate the efficacy of reduced nutrient medium in 2 sources of oocytes. Oocytes were in vitro matured in identical medium either in our laboratory or during shipment from a commercial supplier. Oocytes in our laboratory were derived from feedlot heifers while purchased oocytes were obtained from culled cows. Zygotes were cultured using sequential medium with fraction V BSA. In step 1/step 2, embryos were cultured using 100% (glucose 0.5 mM/fructose 3.0\u2009mM, pyruvate 0.3/0.1\u2009mM, lactate 10.0/6.0\u2009mM, NEEA 1 7/1 7 MEM, EAA 0.25 7/0.5 7 MEM), 25% or 6.25% of standard nutrient concentrations. On Day 3, embryos were moved to step 2 as follows: 100% to 100%, 25% to 25%, 25% to 100%, 6.25% to 25%, or 6.25% to 100%. Lipid content of single mature oocytes from both sources was determined using gas chromatography coupled to an ISQ-LT MS/MS (GC-MS; Thermo Scientific, Waltham, MA, USA). Data (mean\u2009\ub1\u2009s.e.m.) were analysed using ANOVA (P\u2009<\u20090.05). When oocytes from feedlot heifers were used, blastocyst development and cell number did not differ between treatments. When oocytes from culled cows were used, blastocyst development was improved after embryo culture in 25-25% (45.1\u2009\ub1\u20093.3%) and 6.25-25% (46.6\u2009\ub1\u20093.2%) compared with 100-100% (34.2\u2009\ub1\u20093.2%). However, inner cell mass number of blastocysts cultured in 25-25% (25.6\u2009\ub1\u20092.5) and 6.25-25% (26.0\u2009\ub1\u20092.6) was reduced compared with 100-100% (41.4\u2009\ub1\u20094.5); TE and total cell number did not differ. Embryos cultured in 100-100%, 25-100%, and 6.25-100% were equivalent. Metabolomics revealed that 10 lipid compounds (polyunsaturated fatty acids, glycosyldiacylglycerols, and glycerophospholipids) differed in abundance between the two sources of oocytes. These results show that oocytes from different sources lead to different experimental outcomes, likely due to a combination of age, body condition, diet, and hormone treatment of the female. Oocytes from culled cows result in embryos that develop to blastocysts better in a reduced nutrient environment, although these embryos have fewer inner cell masses, suggesting that quality may be reduced. Embryos from feedlot heifer oocytes are relatively immune to nutrient fluctuations. Different endogenous fatty acid reserves in the oocyte may lead to differing metabolic strategies in the subsequent embryo, altering their response to substrate availability during in vitro culture. These results also demonstrate that reduction of nutrients during culture has no detrimental effect on blastocyst development or total cell number in either oocyte source, but that inner cell mass formation requires increased nutrient provision

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