1.1 Background

Cell culture laboratories in general require facilities with very high standards of hygiene and air quality. Maintaining a Grade-A environment (ISO 4 or above) in these laboratories is usually necessary with respect to particle counts and airborne microbial colony forming units. This minimizes the risk of contamination and permits cell culture for prolonged periods of time.

In vitro fertilization (IVF) laboratories also require high standards of cleanliness, but in addition, special requirements add an element of increased complexity. For some aspects, compromises must be made between the most desirable standards and what is practically possible. This refers particularly to the necessity for close proximity to the operating room (OR) where oocyte retrievals and embryo transfers are performed, as well as other outpatient surgery such as testicular biopsies. The sensitive nature of the oocytes and embryos places certain restrictions on ventilation and disinfection. The relatively short culture period in IVF on the other hand allows for somewhat greater flexibility while other aspects place certain restrictions on attainment of Grade-A hygienic standards. This pertains particularly to temperature sensitivity and very low tolerance to toxic compounds, such as volatile organic compounds (VOCs) which, for example, places restrictions on the use of disinfectants such as alcohol (Morbeck 2015; Mortimer 2005).

The aim of this chapter is to describe the facilities and layout of an IVF laboratory and how the requisite quality and hygiene standards are met while conforming to applicable regulations.

The chapter will be organized according to the following sub-headings:

• 
  

  General aspects of air quality

  
  
• 
  

  Hygiene

  
  
• 
  

  Layout of the laboratory and communication with the OR

  
  
• 
  

  Laboratory storage

  
  
• 
  

  Safety in the IVF laboratory

  
  
• 
  

  Security for the IVF laboratory

  
  
• 
  

  Gas and electrical power supply.

1.2 General Aspects of Air Quality in the IVF Laboratory

Human embryo culture is highly sensitive to airborne contaminants – particulate, microbial and organic – and requires high standards of air purity and quality. Therefore, it is necessary to place high demands on facilities for assisted reproductive technology (ART) laboratories to ensure optimal culture conditions. Some countries place strict demands through legislation and formal regulation whereas others work through guidelines. For example, since 2004 the European Union has had a directive in force to ensure high standards in ART laboratories (Hreinsson and Kovačič, 2019). There is also a wide consensus within the ART community that air quality in the IVF laboratory needs to meet certain criteria and that contaminants must be kept to a minimum (Esteves & Bento 2016; Mortimer et al., 2018). For a discussion on ISO classes of air quality vs. the GMP A–D classes, see Guns and Janssens (2019).

In general, the ART laboratory can be compared to an OR in terms of air quality and should be supplied with HEPA (high-efficiency particulate absorption) filtered air to achieve a comparable level of cleanliness. This requires at least ISO class 7 for air quality (see Table 1.1). It is recommended that the laboratory should be located in the middle of the facilities so that the laboratory does not lie adjacent to the outside walls or windows of the building. In essence, this means maintaining the “room within the room” principle, facilitating the maintenance of air purity and decreasing the risk for contamination.

It is well established that VOCs can have a detrimental effect on human embryo culture (Esteves & Bento, 2016). Materials used when building the laboratory, including flooring and wall covering as well as furniture and equipment, should have low off-gassing rates, and the entire laboratory in new or renovated facilities should be allowed a burn-off period of several weeks before being taken into use.

The HVAC system (heating, ventilation and air conditioning) is highly important and usually the laboratory and OR run on a separate, isolated system. Typically the HVAC system is installed during construction since most buildings do not have sufficient capacity in the regular systems. By including carbon and potassium permanganate filters in the ventilation system, toxic VOCs can be absorbed and removed from the air flowing into the laboratory. All filters in the HVAC system must be monitored and maintained regularly with replacements scheduled as necessary. To reduce the burden on these filters, the location of the air intake for the HVAC system must also be considered. Usually these are placed on the top of the building to avoid car exhaust and other contaminants which can be a problem in large cities. In hospitals, helicopter platforms on the roof of the building may also create complications and need to be taken into consideration.

The number of air changes in a room of ISO class 7 should be 60–150 per hour. However, IVF laboratories usually aim at 15–20 air changes per hour with only part of the air flow coming directly from the air intake and the rest being recirculated. This is because too high air flow rates may impact temperature stability of heating zones and in culture dishes and also increase evaporation rates from culture droplets during culture media preparation. Here, a balance must be found between air quality and maintaining general physical parameters within acceptable ranges. Laminar flow hoods are to be used for preparation of the dishes to counteract this potential problem.

Maintaining humidity levels between 40% and 50% in the laboratory is optimal since lower levels will increase microdroplet evaporation whereas higher levels may induce formation of mold. There is also the issue of maintaining a healthy environment for staff. The same applies to temperature in the laboratory which should be kept at a level ensuring a comfortable environment. A high room temperature is not optimal for incubators which are designed to run at a temperature differential of approximately 13–15°C above ambient surroundings.

It is standard for all clean rooms to have a positive pressure differential from the laboratory to their surroundings. This will reduce the risk of contaminants entering the laboratory from the outside. This pertains to general air contaminants as above but is also relevant to the OR since the disinfectants and cleaning agents necessary for optimal patient care must not carry over into the laboratory. Each laboratory needs to be able to perform tests to determine particle counts, microbial contamination, and VOCs in the air in the facilities.

Typically, an ART clinic operates as an outpatient clinic with minor surgery under local anesthesia only, the great majority of patients being ambulatory. Although emergency provisions and access for persons with disabilities should be possible in the facilities, this is usually feasible in office buildings as well. If general anesthesia and more invasive surgery are to be performed in the facilities, this places additional demands on patient monitoring, emergency access and around the clock availability. When planning general anesthesia and more invasive surgery, separate demands must be met. This chapter does not address the issues raised in these cases

1.3 Hygiene

When operating a clean laboratory with adequate ventilation and filtration, it becomes clear that the staff is a major source of particle contamination in the IVF laboratory. Therefore the use of non-shedding clothing and hair covers is mandatory in the IVF laboratory. Gloves are used for personal protection when working with body fluids, such as sperm samples or follicle aspirates. In addition, the use of masks is recommended in certain instances, such as when performing embryo biopsy and tubing for preimplantation genetic testing. Hand hygiene must be observed at all times since use of gloves is often considered inconvenient and possibly risky when working with fine manipulation, as in the IVF laboratory setting. Rings, wrist watches, and jewelry should not be worn in the OR or in the laboratory, as is the general hospital standard. In addition to soap and water, hand disinfectants which do not emit VOCs should be used.

Use of alcohol for disinfection in the laboratory is normally discouraged because of the potential toxic effects associated with it. Although using alcohol on work surfaces in a well-ventilated laboratory after all cell culture work has been performed may be in order, especially when using incubators with closed gas circulation, it is generally avoided. Instead quaternary ammonium compounds which do not emit VOCs can be used, or hydrogen peroxide solutions which do not leave a residue after use. Each laboratory needs to verify and validate the cleaning methods used as well as establishing general hygienic standards, finding a balance between maintaining good hygiene and avoiding infections while minimizing any potential toxic effects to the gametes and embryos.

All work areas must be thoroughly cleaned both in terms of pathogens but also to avoid DNA-contamination in embryology laboratories performing preimplantation genetic testing. Supplies in cardboard boxes must never enter the laboratory (see 1.5 Laboratory Storage). Administrative work should be minimized in the laboratory and OR covers for computers should be considered.

To facilitate cleaning of the rooms, sealed mats with rounded corners should be used for the floor and the ceiling should be sealed with rounded corners as well. Lights should be built into the ceiling. Proper floor material, such as metal sheets, should be used where liquid nitrogen is handled. If windows are exposed to direct sunlight, dark shades need to be applied.

The laboratory and OR should be tested for airborne microorganisms using sedimentation plates or specialized measuring devices to evaluate the standard of the air quality in this respect.

The issue of lighting in the IVF-laboratory often comes up. As a general rule, low intensity lighting is recommended while maintaining a safe and secure working environment is paramount. When considering light sources in the IVF-laboratory, it is useful to consider that oocytes and embryos are kept in incubators most of the time and are not exposed to ambient light. The majority of light exposure to the ovum and embryo occurs during microscopic examination and micromanipulation. The light sources in the microscopes are typically halogen light bulbs from 30 watts up to 100 watts and the operator can minimize this exposure by using the lowest possible intensity required to perform the work (Ottosen et al., 2007). Use of colored light filters can be considered as well.

Ambient light should be reduced and direct sunlight cannot be allowed in the IVF laboratory.