Characterization and comparison of commercial oils used for human embryo culture.

STUDY QUESTION: Are there significant differences between the available commercial oil brands used for human IVF? SUMMARY ANSWER: Important differences have been detected among the tested oil brands in their potential to stabilize culture conditions and, more importantly, in their direct effect on embryo development and viability. WHAT IS KNOWN ALREADY: Mineral oil is a critical component of the human culture system due to its protective and stabilizing roles during in vitro embryo development. Many different oils are available on the market, with differences in their viscosity, density and overall quality. STUDY DESIGN, SIZE, DURATION: Thirteen different commercial oil brands were compared. PARTICIPANTS/MATERIALS, SETTING, METHODS: Each oil was firstly analyzed to assess its viscosity, density, peroxide value and potential oxidation. Secondly, the capacity of each oil to reduce pH, osmolality and temperature fluctuations during embryo culture and manipulation was compared. Lastly, a sensitive mouse embryo assay (MEA) protocol, previously optimized to detect toxicity in oils samples, was used to compare the overall quality of the different brands in terms of embryo developmental rates up to the blastocyst stage. At the end of the MEAs, a triple labeling protocol was applied to analyze Oct4+ cells, apoptotic cells and total cell counts in the blastocysts obtained by fluorescence microscopy. MAIN RESULTS AND THE ROLE OF CHANCE: Significant divergences were detected in the rise of osmolality and the equilibration and stability of pH between different oils, which could be correlated to their physico-chemical characteristics. In particular, oil samples with a higher viscosity tended to offer an additional protection against fluctuations in the culture conditions, however, the differences in temperature stability between oils were minor. Two out of the 13 oil samples, which were commercially available, were identified as embryo-toxic by applying the MEA protocol with increased sensitivity for toxicity detection. Additionally, substantial differences in the total number of cells and the number of cells in the inner cell mass of the obtained blastocysts were also detected between oil groups. LIMITATIONS, REASONS FOR CAUTION: A single lot of oil was used for each brand and, thus, lot-to-lot variations in oil quality could not be determined. However, several bottles from the same oil were included to account for potential intra-lot variability. WIDER IMPLICATIONS OF THE FINDINGS: Commercial oils differ in both their physical characteristics and their performance in maintaining the stability of the culture conditions during in vitro embryo culture. Oil selection is important for embryo culture success. Additionally, the detection of embryo-toxic oils which had already been released to the human IVF market showcases the importance of applying sensitive MEA protocols for a better detection of toxicity in this type of samples. STUDY FUNDING/COMPETING INTEREST(S): This study was privately funded. TRIAL REGISTRATION NUMBER: N/A.

Factors of the human embryo culture system that may affect media evaporation and osmolality.

STUDY QUESTION: Which lab-related factors impact the culture system's capacity to maintain a stable osmolality during human embryo culture? SUMMARY ANSWER: Incubator humidity, the volume of mineral oil, the type of culture media and the design of time-lapse dishes have been identified as important parameters that can cause an impact on media evaporation and consequently osmolality during culture. WHAT IS KNOWN ALREADY: Culture medium is a critical component in human embryo culture. Minimizing its evaporation during culture is an adequate strategy to stabilize osmolality and, as a result, improving culture conditions and clinical outcomes. STUDY DESIGN, SIZE, DURATION: The studied variables included media composition and supplementation; volume of mineral oil; incubator humidification; and the type of dish and incubator used. Additionally, six time-lapse dish models were compared in their ability to prevent evaporation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Dishes were incubated in parallel to analyze osmolality during culture between groups: synthetic oviductal medium enriched with potassium versus human tubal fluid medium; protein versus no protein supplementation; dry versus humid atmosphere; high versus low volume of mineral oil. Additionally, media evaporation was compared between six models of time-lapse dishes with distinct designs, cultured in a joint incubator. Two of them were retested in their corresponding incubator to analyze the dish-incubator fit. Daily osmolality measurements were compared between groups. Linear regression was performed to analyze evaporation rates. MAIN RESULTS AND THE ROLE OF CHANCE: Protein supplementation did not significantly affect evaporation. Contrarily, humidity levels inside the incubators, the volume of mineral oil and the type of culture media, played an important role in osmolality stabilization. The design of time-lapse dishes and their recommended preparation protocol heavily influenced their evaporation rates, which were further altered by each incubator's characteristics. Media with initially high osmolalities had a bigger risk of reaching hypertonic levels during culture. LIMITATIONS, REASONS FOR CAUTION: While numerous, the studied variables are limited and therefore other factors could play a role in osmolality dynamics, as well. Incontrollable atmospheric factors could also result in some variation in the observed results between different centers and laboratories. WIDER IMPLICATIONS OF THE FINDINGS: Published literature has extensively described how hypertonic media may impair embryo development and negatively affect clinical outcomes; therefore, maintaining a stable osmolality during culture should be considered essential. This work is of interest both for embryologists when analyzing their culture system and methodologies, as well as manufacturers in charge of designing IVF consumables. STUDY FUNDING/COMPETING INTEREST(S): This study was privately funded. TRIAL REGISTRATION NUMBER: N/A.

Polyhydroxyalkanoates (PHA) biosynthesis from kraft mill wastewaters: biomass origin and C:N relationship influence.

The goal of this study is to evaluate the feasibility of PHA biosynthesis from kraft mill effluent using the batch system evaluating the biomass origin and C:N relationship influence. To evaluate feasibility, batch assays were carried out. Also, two levels of the BOD5:N:P relationship (100:5:1 and 100:1:0.2) and three different sludge origins were considered. Inocula were obtained from activated sludge treatment plants for a) sewage (SAS), b) paper mill (PAS) and c) kraft (KAS). The results show that the maximum Biological Oxygen Demand (BOD5) and Chemical Oxygen Demand (COD) removal was 80.5% and 59.7% respectively using KAS as inoculum. In these assays, kinetics constants were 17.9±3.2 mg L(-1) and 46.5±1.2 d(-1) for (KCOD) and (rmax), respectively under a BOD5:N:P relationship of 100:5:1. The maximum PHA accumulation was obtained under a BOD5:N:P relationship of 100:1:0.2 on the third day of batch assays using PAS sludge with 25.72% of the cells accumulating PHA and on the fifth day in batch using SAS and KAS sludge with 25.85% and 30.40% of cells accumulating PHA, respectively. Yields obtained for the 100:1:0.2 relationships ranged from 0.10-0.14 mg PHA mg(-1) COD.