2.1 Introduction

Incubators represent the most important piece of equipment in an in vitro fertilization (IVF) laboratory since embryos spend the largest part of their in vitro development within an incubator’s atmosphere. Incubators, together with embryo culture media, are intended to directly and indirectly provide stable physicochemical conditions that best mimic the natural environment in the female reproductive tract. The stability of these conditions significantly influences the success of the IVF program. Modern incubators can be very sophisticated devices that can be upgraded with integrated micro cameras and linked to computer programs. Although the incubator’s technical details may sometimes be difficult to understand, it is important for clinical embryologists to know how to control incubator operation and properly maintain stable physical and hygienic conditions.

When considering investment in IVF laboratory equipment, it makes sense to first think about incubators. The decision about which type of incubator would best meet the requirements of the laboratory is not easy because of the wide range of different types of incubators. This chapter describes the characteristics of the various types of incubators and the related physical and chemical factors affecting embryo development.

2.2 Incubator Types

During the early period of cell culture development, and even later with the first human embryos being cultured in vitro, a glass jar (desiccator) placed into a heating chamber controlled by a thermostat was used as an incubator. Distilled water was poured into the lower part of the jar to ensure a sufficiently high relative humidity in the space. This system was the first “air-jacket” CO₂ incubator, and the first IVF baby was conceived with the help of this type of incubator.¹

During the 1960s, the first commercial CO₂ incubators were developed by New Brunswick Scientific (New Jersey, USA). In 1984, Shel Lab (Cornelius, USA) introduced the general purpose incubator, which had a warm air jacket, heated door, and five integrated heating elements to maintain a uniform temperature, with no hot spots. Gas concentrations were monitored by thermocouple sensors, which were very sensitive to temperature and humidity. Different companies launched many similar types of incubators, almost all with installed fans to enable equal physical conditions in all parts of the chamber.

At the beginning of the 2000s, a new line of CO₂ incubators appeared on the market, featuring a direct-heat and fanless design. These incubators were lighter than water-jacketed incubators and had advanced infrared (IR) CO₂ sensors, working without sensitivity to other physical parameters. The incubator’s interior became a seamless, deep-drawn inner stainless steel chamber. Such simplification of the interior facilitated easier cleaning and prevented contamination. The humidification system was also simplified and water condensation was prevented. They also contained their own LCD screens and on-board diagnostics.

Benchtop incubators were developed during the 1990s. Benchtop models modified for IVF (e.g., FIV series from Carteau, Bagnolet, France) had integrated microplates and a humidification system. This innovation offered optimal temperature uniformity and stability in 4-well culture dishes. The incubators had a simple construction in which multiple incubation chambers were stacked.

In the last decade, the evolution of embryonic culture incubators has been marked by the idea of placing a time-lapse (TL) microcamera inside a classic incubator (Primovision, Vitrolife, Göteborg, Sweden), enabling the continuous monitoring of embryo development. Linking TL cameras to appropriate software has opened up a new dimension in clinical embryology, being able to document the embryo continuously throughout the time of culture (more details on time-lapse in Chapter 10). One of the biggest advantages TL incubators provide is uninterrupted embryo culture as daily embryo scoring can be performed without removing the culture dish from the incubator.

2.2.1 Selection of Incubators

The development of incubators in recent years has made incredible progress. IVF laboratories can hardly adapt to such rapid development of technology, as it is resource demanding to replace all old incubators with new ones overnight. Therefore, in today’s IVF laboratories, it is possible to find all types of incubators (Figures 2.1 and 2.2) with different technical characteristics (Table 2.1).

Figure 2.1 Incubators for overnight or longer embryo culture: a. large-box incubator; b. benchtop incubator; c. benchtop multi-room incubator; d. time-lapse incubator.

Figure 2.2 Holding incubators: a. smaller box incubator for sperm preparation; b. glass jar placed above an opening in the surface of the laminar flow through which CO₂ is introduced; c. closed workstation with controlled incubation atmosphere and integrated microscope.

Table 2.1 Types of incubators for human IVF and their technical characteristics

Type	Technical properties and options
Classic side-door incubators for overnight or longer culture: Large-box incubators Smaller box incubators	– Water jacket – Single door – Heated doors – Stainless steel chamber and shelves – Active humidification – Thermocouple CO2 sensors – Galvanic O2 sensors – Passively circulated air – Single gas port – CO2 or gas mixture – Gas mixing system – O2 control – Air filtration – Air sterilization by UV – Chamber heat sterilization – Chamber disinfection – Display line diagram option – Memorizing values – On-board diagnostics – RS232 port – Alarm	– Air jacket – Multiple doors – Non-heated doors – Cooper chamber and shelves – Passive humidification – Infrared CO2 sensors – Zirconium O2 sensors – Fan circulated air – Double gas ports – CO2 and N2 – No gas mixing system – No O2 control – No air filtration – No UV – No heat sterilization – No disinfection – No line diagram options – No display of history – No diagnostics – No RS232 port – No alarm
Multichamber incubators for overnight or longer culture: Drawer incubators Benchtop incubators Time-lapse benchtop incubators	– Individual chambers – Heated microplates adjusted to specific petri dish brands – General sensor for temperature and heating elements for all compartments – Mixed gas connection; single gas port – Gas filtration – Shared provision of mixed gas into all compartments through one gas line – Dry culture system – Built-in pH measuring system – Display line diagram – Memorizing values – On-board diagnostics – RS232 port – Alarm – For further technical characteristics and options for TL benchtop incubators, see ESHRE recommendation paper.2	– Shared chambers – Non-adjusted microplates to specific Petri dish brands – Individual sensor for temperature and heating elements for each compartment – Integrated gas mixer; separated gas ports – No gas filtration – Provision of mixed gas into each compartment through separated gas lines – Humid culture system – No pH measuring system – No line diagram option – No history – No diagnostics – No RS232 port – No alarm
Holding and/or working incubators for keeping culture media, gametes, embryos in a CO2 culture atmosphere for a short period during handling with oocytes and embryos Smaller box working incubators Lid incubators Combined incubator–workstation	Smaller box incubators, located near or within laminar flow cabinets. Glass jars or metal lids for covering petri dishes, with gas tubing connection or positioned on the gas exit opening on the laminar flow hood heating plate. Hermetically closed combined incubator–workstation with controlled temperature and gases and with integrated microscope.
Other types Sealed bag incubators In vivo incubators Microfluidic incubators	Sealed plastic bags, filled with gas mixture and sunk into a warm water bath (more appropriate for veterinary IVF). In vivo embryo culture or intravaginal embryo culture in closed tubes, permeable for gases from vagina. Closed device connected to the tubing system that enables constant circulation of culture medium around embryos.

Among the available incubators in the laboratory, it is necessary to identify those that will be used for overnight or extended culture of embryos to restrict the frequency of opening, and those that will serve as holding or working incubators. Holding and working incubators are dedicated to the preincubation of culture media and for short-term incubation, e.g., during sperm preparation, oocyte pick-up, fertilization, and cryopreservation.

While using large incubators, it is recommended that petri dishes with biological material from no more than one patient/couple should be kept on each shelf (or material from four to six patients in each incubator). With benchtop incubators, the recommendation is that only one patient’s gametes/embryos should be cultured per incubator compartment.³ In this way, the physical perturbation of the culture environment and the risk of a sample mix-up are reduced to a minimum. Time-lapse incubators and single-step media can enable uninterrupted embryo culture to the blastocyst stage. In addition, continuous monitoring of the morphodynamics of embryonic development and identification of embryo cleavage irregularities are possible, without any opening of the chamber.² Despite the remarkable advances in incubator technology, it is still difficult to claim that certain types provide better clinical results. Several studies have been conducted to compare the laboratory and clinical performance indicators after using large-box and small benchtop incubators for human gamete and embryo culture.⁴ Some differences in embryo development have been observed between two types of incubators in a few studies.^{5, 6} However, critical analysis identified limitations in the study design and when interpreting results with lack of consideration for many confounding variables that make it impossible to identify the effect of the incubator alone (see also Chapter 10). Most other trials demonstrate some differences in the culture parameters, but they could not confirm differences in any endpoint between incubators.^7–9 Nevertheless, from all comparisons made between different types of incubators, it is apparent that smaller compartments provide more rapid recovery of the temperature and gas concentration. This definitively results in less stress for oocytes and embryos, presumably resulting in improved embryo quality, which is also a goal of clinical embryology (see also Chapter 8).

Even though the embryo culture conditions have improved with new incubator types, many ART techniques still require manipulation of oocytes or embryos outside incubators (such as oocyte pick-ups, oocyte denudation, cryopreservation, ICSI, embryo biopsy, artificial blastocoel collapsing). Due to variations in physicochemical parameters during the performance of these techniques, they represent the most sensitive part of the IVF procedure and cause the most stress to oocytes and embryos. Some laboratories avoid this by using smaller working, non-culture box or lid incubators integrated into laminar flow benches. Glass or metal funnels, connected to a mixed gas source (Figure 2.2b), are also practical for maintaining culture conditions by using them to cover dishes during procedures. Even more stable conditions during oocyte and embryo manipulations can be achieved in a closed workstation with regulated temperature and in some cases also regulated gas concentration (Figure 2c). The performance of key IVF procedures, as those mentioned above, in a controlled atmosphere of closed laminar flow hoods can significantly improve clinical results.¹⁰

2.3 Installation

Before a new incubator is used for embryo culture, it must be properly cleaned and properly validated (Figure 2.3). It is recommended that authorized technical staff connect the new incubator to the electrical outlet and to the gas connections and set-up desired values in the menu. During the test run it is also advisable to keep the incubator out of the IVF laboratory, as the installation process, parameter setting, and incubator calibration / validation are expected to be a time-consuming process that may interfere with routine IVF work. In the laboratory, the incubator should be located near the laminar flow hood and connected to a permanent power source (uninterrupted power source, i.e., battery or generator).

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Related posts:

1. Chapter 4 – Embryo Metabolism and What Does the Embryo Need?
2. Chapter 6 – Optimal Handling Techniques for Culture of Human Embryos
3. Chapter 8 – Timing of Embryo Culture
4. Chapter 12 – The Changing Culture of Embryo Culture

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