Ovarian inflammation mediated by Toll-like receptor 4 increased transcripts of maternal effect genes and decreased embryo development†.

Obese women are subfertile and have reduced assisted reproduction success, which may be due to reduced oocyte competence. We hypothesize that consumption of a high-fat/high-sugar diet induces ovarian inflammation, which is a primary contributor to decreased oocyte quality and pre-implantation embryo development. To test this hypothesis, C57BL/6 (B6) mice with a normal inflammatory response and C3H/HeJ (C3H) mice with a dampened inflammatory response due to dysfunctional Toll-like receptor 4 were fed either normal chow or high-fat/high-sugar diet. In both B6 and C3H females, high-fat/high-sugar diet induced excessive adiposity and hyperglycemia compared to normal chow-fed counterparts. Conversely, ovarian CD68 levels and oocyte expression of oxidative stress markers were increased when collected from B6 high-fat/high-sugar but not C3H high-fat/high-sugar mice. Following in vitro fertilization of in vivo matured oocytes, blastocyst development was decreased in B6-high-fat/high-sugar but not C3H high-fat/high-sugar mice. Expression of cumulus cell markers of oocyte quality were altered in both B6 high-fat/high-sugar and C3H high-fat/high-sugar. However, there were no diet-dependent differences in spindle abnormalities in either B6 or C3H mice, suggesting potential defects in cytoplasmic maturation. Indeed, there were significant increases in the abundance of maternal effect gene mRNAs in oocytes from only B6 high-fat/high-sugar mice. These differentially expressed genes encode proteins of the subcortical maternal complex and associated with mRNA metabolism and epigenetic modifications. These genes regulate maternal mRNA degradation at oocyte maturation, mRNA clearance at the zygotic genome activation, and methylation of imprinted genes suggesting a mechanism by which inflammation induced oxidative stress impairs embryo development.

Fatty acids present in commercial albumin preparations differentially affect development of murine embryos before and during implantation.

OBJECTIVE: To characterize fatty acid (FA) profile of commercially available albumin products and determine their effect on embryonic development. DESIGN: Research study. SETTING: Private research facility. ANIMAL(S): Outbred mice aged 4-8 weeks. INTERVENTION(S): Gas chromatography-mass spectrometry was used to quantify the FA content of 15 commercial albumins. Embryos were produced in media containing different albumin products, with or without carnitine or exogenous FA supplementation, to determine their effect on embryo development in vitro. MAIN OUTCOME MEASURE(S): Total micrograms of FA per milligram of albumin for the 15 albumin products, blastocyst development, cell number, allocation to the trophectoderm (TE) or inner cell mass (ICM), and evaluation of morphology during implantation. RESULT(S): The albumin products contained 0.07-16.77 µg total FA/mg albumin. Compared to media with with >1.4 µg FA/mg albumin, media with <0.5 µg FA/mg albumin supported improved blastocyst development, and addition of carnitine mitigated this difference. Addition of palmitoleic acid or oleic acid individually did not improve blastocyst development and decreased ICM:TE ratio. However, in the presence of carnitine, there was improved blastocyst development and maintenance of the ICM:TE ratio. Embryos cultured in Vitrolife human serum albumin with supplementation of carnitine, palmitoleic acid, and oleic acid were more likely to develop cells positive for POU5F1 in an extended embryo culture than embryos cultured in Origio serum protein substitute. CONCLUSION(S): Commercial albumin products contain FAs, which vary in abundance. These FAs have different effects on embryo development and quality before and during the implantation stage. Several of these albumin preparations are routinely used for human-assisted reproductive technologies; therefore, serious consideration is warranted when selecting a product for clinical use.

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 + 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.

Egg cylinder development during in vitro extended embryo culture predicts the post transfer developmental potential of mouse blastocysts.

PURPOSE: To establish parameters during mouse extended embryo culture that accurately predict fetal developmental potential of a blastocyst without performing embryo transfer. METHODS: Embryos of three varying qualities were produced: poor quality embryos produced from in vitro matured oocytes (IVM), intermediate quality embryos produced from in vivo matured oocytes followed by in vitro fertilization and embryo culture (IVF); high quality embryos developed in vivo (VIVO). Embryonic day (E) 3.5 embryos from each group with similar morphologies were used for surgical embryo transfer to assess implantation and fetal developmental potential, in addition to placing these embryos into extended culture until E 8.5 to examine outgrowth area, egg cylinder volume, epiblast cell number, and outgrowth morphologies by immunofluorescence and 3D confocal microscopy. RESULTS: The proportional differences in epiblast cell number are strikingly similar to fetal development following embryo transfer, suggesting that this parameter may be indicative of the potential of an embryo to successfully develop into a fetus. CONCLUSION: Extended embryo culture provides more accurate information regarding developmental potential than blastocyst morphological assessment. Specifically, epiblast cell number is an accurate and valuable predictor of fetal developmental potential. This work sets the stage for routine evaluation of embryo quality past the time embryos would normally be transferred. The ability to determine post implantation potential without embryo transfer may greatly improve efforts to culture higher quality embryos in vitro for human IVF, as well as reducing animal use and eliminating confounding maternal factors associated with embryo transfer experiments in research.

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.

A carnivore embryo's perspective on essential amino acids and ammonium in culture medium: effects on the development of feline embryos.

Carnivores are an interesting model for studies of embryonic amino acid metabolism and ammonium (NH4+) toxicity given the high-protein content of their diets. Our objectives were to examine concentration- and stage-specific effects of essential amino acids (EAA; 0×, 0.125×, 0.25×, 0.5×, or 1.0× the concentrations in Minimum Essential Medium) and NH4+ (0, 300, or 600 µM) on the development and metabolism of feline embryos. The presence of EAA, regardless of concentration, during days 3-7 of culture increased (P < 0.01) the proportion of embryos that initiated hatching (>14.3%) and the total number of cells per blastocyst (>148.3 cells) compared to embryos cultured without EAA (0.0% and 113.2 ± 3.7 cells, respectively). The presence of EAA during days 1-3 (0.25×) and 3-7 (1.0×) of culture increased (P < 0.01) the proportions of embryos that formed blastocysts (82.9 ± 4.2%) and initiated hatching (32.9 ± 5.2%), and the number of cells per blastocyst (247.9 ± 12.1 cells), compared to control embryos (60.0 ± 5.3%, 0.0%, 123.2 ± 8.1 cells, respectively). The presence of NH4+ in the medium did not affect (P > 0.05) development of feline embryos. The addition of EAA or NH4+ during culture did not affect (P > 0.05) the production of Gln by feline embryos, but decreased (P < 0.05) production of Ala and increased (P < 0.05) production of urea. Additional work is needed to determine if our observations are unique to feline embryos or reflect an adaptation to a high-protein diet that is conserved in other carnivores.

Exogenous growth factors do not affect the development of individually cultured murine embryos.

PURPOSE: The objective of this study was to evaluate the effects of multiple growth factors on the development of individually cultured murine embryos. METHODS: Embryos produced by in vitro fertilization using in vitro (IVM) or in vivo (IVO) matured oocytes from three strains of mice (CF1, Swiss Webster, B6D2F1) were cultured individually (10 µl) in the absence (control) or presence of growth factors (paf, epidermal growth factor [EGF], insulin-like growth factor 1 [IGF-1], and granulocyte-macrophage colony-stimulating factor [GM-CSF]). Blastocyst formation, hatching, and blastocyst cell numbers (trophectoderm, inner cell mass, and total) were evaluated on days 4 and 5 of culture. Post-hatching development of CF1 IVO embryos was also evaluated in vitro and in vivo. RESULTS: The presence of growth factors did not improve the proportion of embryos forming blastocysts or initiating hatching for any of the types of embryos tested. The only significant (P < 0.05) effect of growth factors was a decrease in the proportion of embryos that formed blastocysts by day 5 in CF1 IVM embryos. The presence of growth factors also did not affect blastocyst cell numbers. For CF1 IVO embryos, the presence of growth factors during culture did not affect the proportion of embryos that attached to fibronectin-coated dishes, the size of the resulting outgrowths, or in vivo development following transfer. CONCLUSION: Combinations of paf, EGF, GM-CSF, and IGF-1 did not improve development of murine embryos cultured individually in a sequential medium containing a defined protein source.