The effects of chemical and physical factors on mammalian embryo culture and their importance for the practice of assisted human reproduction.

BACKGROUND: Although laboratory procedures, along with culture media formulations, have improved over the past two decades, the issue remains that human IVF is performed in vitro (literally 'in glass'). METHODS: Using PubMed, electronic searches were performed using keywords from a list of chemical and physical factors with no limits placed on time. Examples of keywords include oxygen, ammonium, volatile organics, temperature, pH, oil overlays and incubation volume/embryo density. Available clinical and scientific evidence surrounding physical and chemical factors have been assessed and presented here. RESULTS AND CONCLUSIONS: Development of the embryo outside the body means that it is constantly exposed to stresses that it would not experience in vivo. Sources of stress on the human embryo include identified factors such as pH and temperature shifts, exposure to atmospheric (20%) oxygen and the build-up of toxins in the media due to the static nature of culture. However, there are other sources of stress not typically considered, such as the act of pipetting itself, or the release of organic compounds from the very tissue culture ware upon which the embryo develops. Further, when more than one stress is present in the laboratory, there is evidence that negative synergies can result, culminating in significant trauma to the developing embryo. It is evident that embryos are sensitive to both chemical and physical signals within their microenvironment, and that these factors play a significant role in influencing development and events post transfer. From the viewpoint of assisted human reproduction, a major concern with chemical and physical factors lies in their adverse effects on the viability of embryos, and their long-term effects on the fetus, even as a result of a relatively brief exposure. This review presents data on the adverse effects of chemical and physical factors on mammalian embryos and the importance of identifying, and thereby minimizing, them in the practice of human IVF. Hence, optimizing the in vitro environment involves far more than improving culture media formulations.

Oxygen affects the ability of mouse blastocysts to regulate ammonium.

During embryo culture, ammonium is generated by amino acid metabolism and from the spontaneous deamination of amino acids at 37°C. Although ammonium has been shown to be embryo toxic, few studies have investigated the mechanism(s) by which the early embryo can regulate ammonium. Whilst 20% oxygen represents a source of stress to the developing embryo, it is not known how oxygen affects the physiology of the embryo in the presence of other sources of stress. The aim of this study was, therefore, to investigate possible pathways involved in ammonium sequestration in the preimplantation embryo and the effect of oxygen on the regulation of these pathways. Glutamine and alanine were investigated as possible ammonium sequestration pathways. Amino acid utilization by blastocysts was determined after culture from the postcompaction stage with 0, 150, or 300 µM ammonium (in either 5% or 20% oxygen) and with or without 500 µM L-methionine sulfoximine (MSO), an inhibitor of glutamine synthetase. In the presence of MSO, ammonium production was significantly increased and glutamate was no longer consumed. Glutamine synthetase inhibition with MSO significantly decreased glutamine formation. Ammonium and oxygen independently altered overall amino acid turnover. Together, 5% oxygen and ammonium promoted glutamine production, whereas in the presence of 20% oxygen and ammonium, glutamine was consumed. Data reveal that both oxygen and ammonium affect amino acid utilization by the developing embryo, however, 20% oxygen appears to have the greater impact. Mouse blastocysts can alleviate ammonium stress by its transamination to both glutamine and alanine, but only under physiological conditions.

Analysis of metabolism to select viable human embryos for transfer.

As we move to reducing the number of embryos transferred in a given IVF cycle, ideally down to one, there is an ever-increasing need for noninvasive quantitative markers of embryo viability. Although stage-specific morphologic markers and grading systems have been developed, such an approach is unable to assess the physiological status of the embryo. Analysis of metabolism has proved to be a valuable marker of embryo viability after transfer in animal models. We therefore reviewed what is known about human embryo metabolism, how media systems can affect the patterns of nutrient utilization and the activities of metabolic pathways, and how this relates to the developmental competence of the embryo. It is proposed that a unifying hypothesis of metabolism for the entire preimplantation period is not realistic, given the dramatic changes in embryo physiology that occur from fertilization to blastocyst development, and that the concept of a "quiet metabolism" can be interpreted as stress induced by the presence of high oxygen in the embryo culture/analysis system. Further research is required to fully understand the origins of metabolic stress in embryos for it to be alleviated and to develop a comprehensive range of markers that not only reflect embryo viability, but also sex-specific differences in physiology.

Oxygen regulates amino acid turnover and carbohydrate uptake during the preimplantation period of mouse embryo development.

Oxygen is a powerful regulator of preimplantation embryo development, affecting gene expression, the proteome, and energy metabolism. Even a transient exposure to atmospheric oxygen can have a negative impact on embryo development, which is greatest prior to compaction, and subsequent postcompaction culture at low oxygen cannot alleviate this damage. In spite of this evidence, the majority of human in vitro fertilization is still performed at atmospheric oxygen. One of the physiological parameters shown to be affected by the relative oxygen concentration, carbohydrate metabolism, is linked to the ability of the mammalian embryo to develop in culture and remain viable after transfer. The aim of this study was, therefore, to determine the effect of oxygen concentration on the ability of mouse embryos to utilize both amino acids and carbohydrates both before and after compaction. Metabolomic and fluorometric analysis of embryo culture media revealed that when embryos were exposed to atmospheric oxygen during the cleavage stages, they exhibited significantly greater amino acid utilization and pyruvate uptake than when cultured under 5% oxygen. In contrast, postcompaction embryos cultured in atmospheric oxygen showed significantly lower mean amino acid utilization and glucose uptake. These metabolic changes correlated with developmental compromise because embryos grown in atmospheric oxygen at all stages showed significantly lower blastocyst formation and proliferation. These findings confirm the need to consider both embryo development and metabolism in establishing optimal human embryo growth conditions and prognostic markers of viability, and further highlight the impact of oxygen on such vital parameters.

Glucose consumption of single post-compaction human embryos is predictive of embryo sex and live birth outcome.

BACKGROUND: The aim of this study was to determine the relationship between nutrient utilization by the human embryo and its subsequent viability after transfer. METHODS The embryos of 50 patients having single blastocyst transfer were cultured individually from Day 3 in 10 µl drops of medium G2 under Ovoil in 5%O(2), 6%CO(2), 89%N(2). Patient inclusion in the study was maternal age ≤ 38. Embryos were moved to fresh drops of medium every 24 h. Spent media samples, including controls containing no embryo, were coded, frozen and subsequently analysed blind. Analysis of glucose was performed by microfluorimetry. The sex of children born was recorded. RESULTS: Clinical pregnancy and live birth rates were 58 and 56%, respectively. Glucose consumption by embryos which resulted in a pregnancy was significantly higher on both Day 4 and Day 5 than that by embryos which failed to develop post-transfer (P < 0.01). Furthermore, on Day 4 female embryos consumed 28% more glucose compared with males (P < 0.05). Glucose uptake was independent of embryo grade. CONCLUSIONS: The rapid screening of glucose metabolism by the human embryo on Day 4 and 5 may prove to be a useful metric in the development of algorithms for the selection of embryos for transfer in human IVF. Also, the observed sex-related metabolic difference provides preliminary data to support the hypothesis that male and female human embryos differ in their physiology due to the presence of two active X chromosomes and an altered proteome for a finite time during the preimplantation period.