19.3.1 Air Quality

Poor laboratory air quality is a recognized hazard to the culture of human embryos. While little is known about the actual components that effect these changes, Cohen et al. have postulated that four different categories of pollutants are involved: volatile organic compounds (VOCs); small inorganic molecules such as N₂O, SO₂ and CO; substances derived from building materials (i.e., adhesives and floor tiles); and other polluting compounds (i.e., pesticides, aerosols) [1].

In order to avoid the negative effects of poor air quality, careful consideration should be given to the site and the location of human IVF laboratory. In addition, an appropriate design of the laboratory is essential. The use of the lowest VOC emitting products for all construction materials is recommended. Positive pressure airflow in the laboratory coupled with the use of air purification systems and appropriate in-line filters for incubators will help to minimize the level of airborne contaminants and improve the outcomes. Furthermore, laboratory personnel can also introduce VOCs in the form of perfumes and deodorants; hence, discretion is required [2]. Finally, the use of oil overlay may be an effective way of limiting the impact of any diverse environmental factors.

19.3.2 Light

In IVF lab, embryos are exposed to both microscope and ambient light. Visible light has been shown to be an additional stress and has a deleterious effect on mammalian gamete and embryo development in vitro [3]. It is prudent to work in low illumination and to minimize the amount of time and observation made on gametes and embryos under the microscope. Light can also degrade the integrity of tissue culture media, so ideally media should be kept in the dark.

19.3.3 Temperature

Temperature is another variable of the culture system that impacts various aspects of gamete and embryo function, most notably meiotic spindle stability [4] and possibly embryo metabolism [5]. Maintaining temperature stability around 37 °C is important for the oocyte, followed by the cleavage stage embryo, with increased thermo-tolerance increasing after compaction [2]. Most laboratories set their equipment to run at 37 °C. Using an incubator with stringent temperature control and recovery is important. In addition, avoiding overuse of the incubators is critical to prevent temperature variations. Ambient air temperature can affect how temperature is maintained when embryos are outside of the incubator. Microscope stage warmers and incubator chambers also differ in their ability to hold the temperatures constant at 37 °C. So laboratories should set their thermostats to 71 °F and above and use solid-viewing surface microscope stages with a temperature of >37.5 °C on the warming stage.

19.3.4 pH and Carbon Dioxide (CO₂)

In mammals, the pH shifts from an alkaline environment in the oviduct (7.60 ± 0.01) to an acidic environment in the uterus (6.96 ± 0.01) [6]. Although embryos can develop over a range of media pH, it appears that pH set at or near 7.3 may provide adequate conditions for embryo growth in a static laboratory environment [7–9]. Media pH is primarily determined by the bicarbonate concentration in the media and the CO₂ concentration of the culture incubator. Therefore, it is advisable to use a CO₂ concentration of between 6% and 7% to yield a media pH of around 7.3. To properly monitor CO₂ level, digital CO₂ analysis units are preferred over a liquid-based system such as Fyrite. pH shows a dynamic pattern and is also influenced by specific media components such as lactate, pyruvate, and amino acids. Thus, directly assessing the pH of culture media within a laboratory’s own culture is prudent as part of a rigorous quality control program. A simple and reliable method of checking the pH of a medium is to use color standards; however, this requires the presence of phenol red in the medium. Use of a pH meter is generally considered a more accurate mean of assessing pH. The analysis of media pH is best performed with a blood gas analyzer or with an optical device inside the incubator.

19.3.5 Oxygen (O₂)

Although the atmospheric level of O₂ at sea level is approximately 20.9%, physiological O₂ levels are lower; they vary in different parts of the reproductive tract, typically from 2% to 8% [10, 11]. Human embryo culture has traditionally used an atmosphere of around 20% O₂. More recently, increasing evidence suggests that culture in a low O₂ concentration improves preimplantation embryo development, implantation, and pregnancy rate [12, 13]. Of note, the vast majority of studies on lower O₂ levels for embryo culture have focused on 5%. However, the optimum O₂ concentration for human embryo development has yet to be elucidated, and further, it is unclear whether stage-specific differences exist [2].

19.3.6 Incubator

Incubator selection and management is critical for the success of an IVF program. In considering the type of incubator to be used, the overriding aim is to minimize disturbance to the embryos’ environment and conditions, specifically temperature and pH. There are many types of incubators available for human IVF embryo culture. Box-type incubators have been long used for clinical IVF and were later adapted to smaller box-type incubators. More recently, mini-benchtop incubators have been developed. Such incubators allow for direct heat between the chamber and culture vessel and a direct flow of premixed gas and therefore minimize changes in temperature and pH. Most recently, such chambers have evolved to include time-lapse capability, facilitating the constant monitoring of embryos without interrupted embryo culture [14]. To date, there is no clear consensus as to a superior incubator type, although the efficacy and environmental stability rely heavily on incubator use and management. This reinforces the need for strict quality control as well as proper management of laboratory IVF incubators to optimize their functions and maximize outcomes.

19.3.7 Culture Media

The media that are used to culture human preimplantation embryos is an important factor for the success rates of IVF/ICSI (intracytoplasmic sperm injection). In the first decade of human IVF, the culture media ranged from simple salt solutions such as Earle’s or Tyrode’s solutions to complex media intended for tissue culture such as HamF10. All of them were usually supplemented with different sources of proteins, mostly with fetal or maternal serum. Nowadays, as the industry expanded, numerous commercially culture media are available that contain various components including salts, energy substrates, serum supplements, amino acids, buffer solutions, antibiotics, vitamins, nucleotides, growth factors, and others. Such media can be used either as a sequential system, with different compositions for days 1–3 and 3–6, or as a single medium, used for the whole culture period [15]. Today, with the advent of time-lapse microscopy, the media designed specifically for the purpose of uninterrupted embryo culture have been shown to be effective [16]. Despite all these changes in culture media, it is still unclear whether the composition of the media affects embryo quality and implantation rates and which culture media leads to the best IVF/ICSI success rates [17]. With technological improvements and new approaches to assess embryo metabolism, morphokinetics, and other means of viability assessment, there remains the possibility that media formulations may still be further refined and improved to benefit embryo development and clinical outcomes.

19.3.8 Embryo Density

The number of embryos cultured per drop must also be considered. During IVF, embryos can be cultured either singly or in groups. Culturing embryos singly allows the history of each embryo to be traced. The benefit of group culture is that embryo produces autocrine or paracrine factors that promote development of both itself and surrounding embryos. Culture of embryos in small volumes enables these factors to reach sufficient concentrations to have an impact. Several studies indicate that extended group culture of embryos may be beneficial for human preimplantation embryo development and that the number of embryos per drop and/or incubation volume seems to be an important factor in determining IVF outcome [18, 19].