Abstract
In the armory of medical technology available for alleviation of disease and quality of life enhancement, there is nothing to match the unique contribution of assisted reproductive technology (ART). There is no other life experience that matches the birth of a baby in significance and importance. The responsibility of nurturing and watching children grow and develop alters the appreciation of life and health, with a resulting long-term impact upon individuals, families and, ultimately, society. Thus, the combination of oocyte and sperm to create an embryo with the potential to develop into a unique individual cannot be regarded lightly, as merely another form of invasive medical technology, but must be treated with the respect and responsibility merited by the most fundamental areas of human life.
Introduction
In the armory of medical technology available for alleviation of disease and quality of life enhancement, there is nothing to match the unique contribution of assisted reproductive technology (ART). There is no other life experience that matches the birth of a baby in significance and importance. The responsibility of nurturing and watching children grow and develop alters the appreciation of life and health, with a resulting long-term impact upon individuals, families and, ultimately, society. Thus, the combination of oocyte and sperm to create an embryo with the potential to develop into a unique individual cannot be regarded lightly, as merely another form of invasive medical technology, but must be treated with the respect and responsibility merited by the most fundamental areas of human life.
Successful assisted reproduction involves the careful coordination of both a medical and a scientific approach for each couple undertaking a treatment cycle, with close collaboration between doctors, scientists, nurses and counselors. Only meticulous attention to detail at every step of each patient’s treatment can optimize their chance of delivering a healthy baby. Appropriate patient selection, ovarian stimulation, monitoring and timing of oocyte retrieval should provide the IVF laboratory with viable gametes capable of producing healthy embryos. It is the responsibility of the IVF laboratory to ensure a stable, nontoxic, pathogen-free environment with optimum parameters for oocyte fertilization and embryo development. The first part of this book reveals the complexity of variables involved in assuring successful fertilization and embryo development, together with the fascinating and elegant systems of control that have been elucidated at the molecular level. An increased understanding of basic pathophysiology of disease has revealed that events very early in development may have an impact on health, predisposing children to health risks. Preimplantation embryos are regularly exposed to potential environmental stress during in-vitro manipulations, and this has the capacity to impact later development via epigenetic alterations of the genome prior to establishment of the first cell lineages.
It is essential for the clinical biologist to be aware that the control mechanisms involved in human IVF are complex, and exquisitely sensitive to even apparently minor changes in the environment of gametes and embryos. Temperature and pH are of crucial importance, and many other factors can potentially affect cells at the molecular level. Multiple variables are involved, and the basic science of each step must be carefully controlled, while allowing for individual variation between patients and between treatment cycles. New technologies and strategies continue to be introduced that can influence the risks associated with in-vitro culture; the success of any new innovations in technique and technology can only be gauged by comparison with a standard of efficient and reproducible established procedures. The IVF laboratory therefore has a duty and responsibility to ensure that all of the components and elements involved are strictly controlled and regulated via an effective system of quality management, with all procedures carried out by its most valuable and critical asset: a team of highly trained and responsible professional personnel (Guo 2015; Elder et al., 2015; also see Chapter 9).
Setting Up a Laboratory: Design, Equipment and Facilities
The design of an IVF laboratory should provide a distraction- and risk-free environment in which uninterrupted concentrated attention can be comfortably and safely dedicated to each manipulation, with sensible and logical planning of workstations that are practical and easy to clean. Priority must be given to minimizing the potential for introducing infection or contamination from any source, and therefore the tissue culture area must provide aseptic facilities for safe manipulation of gametes and embryos, allowing for the highest standards of sterile technique; floors, surfaces and components must be easy to clean on a daily basis. The space should be designated as a restricted access area, with facilities for changing into clean operating theater dress and shoes before entry.
Consultation: history, examination, investigations, counseling, consent(s)
Drug scheduling regimen: GnRH agonist pituitary downregulation or oral contraceptive pill to schedule withdrawal bleed
Baseline assessment at start of treatment cycle
Gonadotropin stimulation
Follicular phase monitoring, ultrasound/endocrinology
Induction of ovulation
Oocyte retrieval (OCR)
In-vitro fertilization/ICSI
Embryo transfer
Supernumerary embryo cryopreservation
Luteal phase support
Day 15–18 pregnancy test
Ultrasound assessment to confirm gestational sac/fetal heartbeat
Laboratory Space Layout
The range of treatment types to be offered, the number of cycles per year and the manner in which the cycles will be managed all dictate the appropriate layout design and the equipment and supplies required. Four separate areas of work should be equipped according to need, arranged and set up to accommodate the flow of work according to the sequence of procedures in an IVF cycle:
1. Andrology: semen assessment and sperm preparation; surgical sperm retrieval
2. Embryology: oocyte retrieval, fertilization, embryo culture and transfer
3. Cryopreservation: sperm, oocytes, embryos, ovarian and testicular tissue
4. Micromanipulation: ICSI, assisted hatching, biopsy procedures.
Careful consideration should be given to the physical maneuvers involved, ensuring ease and safety of movement between areas to minimize the possibility of accidents. Bench height, adjustable chairs, microscope eye height and efficient use of space and surfaces all contribute to a working environment that minimizes distraction and fatigue. The location of storage areas and equipment such as incubators and centrifuges should be logically planned for efficiency and safety within each working area; the use of mobile laboratory components allows flexibility to meet changing requirements. Many IVF laboratories are now designed with curved joins between walls, floors and ceilings to ensure that no dust settles. For optimal cleanliness on reaching the entrance of the laboratory, two-stage entrance/exits can be incorporated into the changing room areas, so that outdoor clothing is removed at the external section, and scrubs/protective clothing donned at the second stage. Hand-washing facilities must be available in the changing area; sinks should be avoided in culture area, as they can act as a source of microbial contamination.
Light Exposure during ART Procedures
In the course of normal physiology, gametes and embryos exist in a dark environment, and therefore exposure to light is not a ‘natural’ situation. The potential of introducing metabolic stress through light exposure was taken into consideration during the first trials of human IVF in Oldham and Cambridge: dissecting microscopes were fitted with green filters, background lighting in the laboratory was kept low, and during the time of the embryo transfer procedure the lights were extinguished in the operating theater until the embryo transfer catheter had been safely handed over to the physician. Increasingly sophisticated technology in IVF has added the use of more powerful microscopy, with high-intensity light sources. Some spectra of light are known to be associated with generation of reactive oxygen species (ROS), and further data has accumulated about the harmful effects of ROS in IVF. The effects of light exposure in IVF procedures has been studied in detail, with the following conclusions and suggested guidelines (see Pomeroy & Reed, 2015 for review):
Certain wavelengths of light are potentially harmful in ART, and the extent of the damage is related to the duration of exposure, wavelength and intensity. Wavelengths <300 nm UV are absorbed by plastic; near UV-wavelengths of 300–400 nm are associated with increased apoptosis in mouse blastocysts.
Although ambient light is not a significant hazard, cool fluorescent light, as commonly used in laboratories, produces a higher level of ROS and apoptosis in mouse and hamster zygotes than does warm fluorescent light or sunlight (Takenaka et al., 2007).
Embryology laboratories should not be located in areas where direct sunlight might cause damage.
Biological safety cabinet hood lights, ambient lights, headlamps and microscope lamps should be chosen and used with care and attention.
Ninety-five percent of radiation exposure is from microscopes, and the use of green bypass filters may be prudent when viewing gametes and embryos under the microscope.
Laboratory Equipment
Equipment should be selected based on its suitability for the intended purpose, capacity for the intended workload, ease of use and maintenance, availability of service and repair contracts to quality standards, and validation of evidence regarding its correct function. All equipment used in clinical treatments must be of the highest standard available, validated for the intended use, safety checked and properly calibrated, regularly cleaned and maintained. Temperature and gas levels must be strictly monitored and recorded.
Service contracts should be set up with reliable companies, who must provide calibration certificates for the machines used to service and calibrate equipment in the IVF laboratory. Provision must be made for emergency call-out, with alarms fitted to all vital equipment and back-up machines held in reserve. Essential equipment that requires contracts for service and calibration includes:
Incubators
Air filtration equipment
Flow cabinets
Microscopes
Heated surfaces/microscope stages
ICSI/micromanipulation workstation
Centrifuges
Refrigerators and freezers
Embryo/oocyte freezing machines
Osmometers
Liquid nitrogen storage dewars
Electronic witnessing system
Ultrapure water system.
A camera and monitor system that can display and record images is also recommended as part of the basic laboratory set up, for teaching, assessment and record keeping.
Incubators
Carefully calibrated and accurately monitored CO2 incubators that are capable of controlling multiple environmental variables (gas, temperature, humidity) are critical to successful IVF: probably the laboratory’s most important piece of equipment, since embryo development is compromised by environmental fluctuations. The choice of ‘best’ incubator varies between laboratories, depending on workflow, total cycle volume and timeframe of when the cycles are performed. Results can vary between incubators in the same laboratory, and optimal function can only be maintained by strict quality control and correct management that considers the daily patient caseload. Patient samples should be distributed to avoid overuse of any particular incubator, as repeated opening/closing jeopardizes the stability of the culture environment.
The choice of a humidified or nonhumidified incubator depends upon the type of tissue culture system used: whereas humidity is required for standard four-well ‘open’ culture, the use of an equilibrated humidified overlay of clinical grade mineral oil allows the use of incubators without humidity, although this may depend upon the number of days of continuous culture. Dry incubators carry less risk of fungal contamination, are easier to clean and may benefit from the use of thermocouple CO2 sensors, which are not sensitive to humidity.
Traditional stand-alone incubators can accommodate culture dishes for many different patients in a secure, well-insulated environment. Newer models deliver a range of gas concentrations, with CO2 concentrations of 5–6% and variable N2 levels to reduce oxygen concentrations from ambient 21% to as low as 5% (Meintjes et al., 2009). The larger models may incorporate a water-jacket or air-jacket in the door for additional insulation, and may or may not include an internal fan for air circulation. They can be run dry or at 95% relative humidity when water is added to the tray at the bottom of the chamber. A water-jacket not only helps to maintain a consistent temperature under normal circumstances, but also in the event of a power failure. However, the units are heavy, tend to have higher power consumption and have the accompanying risk of adding a source of potential contamination. Air-jacketed incubators warm up quickly, but do not retain heat for long periods without power; they have the benefit of being compatible with heat-sterilizing decontamination methods. Inner gas-tight split doors are essential in order to minimize recovery time after door opening, and CO2 levels must be regularly (preferably continuously) monitored using an infrared CO2 sensor. A single large incubator should not be used to house more than 12 cases at a time.
Smaller benchtop models are portable and therefore more flexible, with pre-mixed gas and sealed gas-tight chambers so that the dishes for each patient can be isolated. These mini-incubators heat the culture dish by direct contact with a warmed surface and are less prone to variation in temperature and pH. Their use excludes the use of laboratory air in the gas mix, and they do not need constant CO2 calibration with external monitoring devices. However, it is extremely important to find a reliable source for the gas mixture, both in the accuracy of the percentages of each gas and in their purity. In addition, the culture system will lose heat rapidly if power is interrupted. In that respect, all incubators should have a back-up battery or generator to protect the power supply in case of power failure.
Large incubators can also be used to equilibrate tubes and bottles of media; this is not possible when only mini-incubators are used, so that HEPES-buffered media might be required for the majority of preparatory procedures. Large incubators also have the advantage of allowing independent probes to be installed, with failure alarms that can operate remotely. Some also have fitted HEPA-volatile organic compound (VOC) filters.
Incubators must be regularly monitored, and readings of the LED display checked and calibrated against independent recordings of temperature and pH monitored by probes placed in a standard ‘test’ culture system. Temperature stability can be monitored with 24-hour thermocouple readings as part of the standard maintenance schedule. There should be a schedule to ensure regular dismantling and cleaning, and a yearly inspection and general servicing by the supplier is recommended. Repeated opening and closing of the incubator affects the stability of the tissue culture environment, and the use of an accessory small benchtop mini-incubator during oocyte retrievals and manipulations helps to minimize disturbance of the larger incubators. Excessive opening/closing can also be avoided by using ‘holding’ incubators for transient procedures such as dish equilibration, sperm preparation methods and brief culture of thawed embryos prior to same day transfer
Irrespective of the type used, in all stand-alone incubators the dishes must be removed from the controlled environment for each stage of manipulation, exposing gametes or embryos to suboptimal temperatures and changes in pH due to reduced CO2 concentration. A number of newer incubators are now available that overcome this problem by incorporating a microscope and imaging system for continuous monitoring of embryo development using time-lapse photography of individual embryos.
Choice of incubator is a critical decision for every IVF laboratory; a distinct advantage of any specific type of incubator has not been clearly demonstrated in terms of human embryo development or clinical outcomes, and selection must be individualized for every laboratory situation. A mix of incubator types helps in covering multiple scenarios, and maintains different options for their use, including implementing new technologies as these evolve. Practical issues such as cost, space, low-O2 capability, gas recovery times and ease of quality management and maintenance will always be important considerations (see Swain, 2014 and Elder et al., 2015 for detailed reviews).
Electronic Witnessing Systems
Sample mismatch, where patients are not correctly linked with their own specimens or treatments, is a catastrophic event in an IVF laboratory, with serious consequences both for patients and staff. Critical points where mismatching of gametes and embryos is most likely to occur include initial gamete collection, mixing of gametes by IVF or ICSI, transfer of gametes or embryos between tubes or dishes, freeze–thaw procedures, and intrauterine insemination or embryo transfer procedures. Adverse events involving sample misidentification invariably receive wide media attention, and have led to the implementation of specific double-checking safety protocols: double-checking of IVF clinical and laboratory protocols is now mandatory. After incidents reported in 2002, the UK regulatory authority (Human Fertilization and Embryology Authority, HFEA) introduced double-witnessing as a requirement in 2006. Double-checking protocols can be difficult to implement because of staffing implications, and are still subject to human error: an embryologist who is continuously distracted by the demands of double-witnessing is vulnerable to errors due to attention lapse/omitting key steps. Electronic witnessing systems have been developed in order to provide safer mechanisms to prevent mix-ups at every stage in the ART procedure. Systems are now available that provide electronic identity checking/witnessing, using either barcode readers or radio frequency identification (RFID), and their use is rapidly extending to fertility clinics worldwide. RFID systems use labels that are attached to all labware, allowing sperm and oocytes to be tracked throughout the IVF process. This technology reduces the chance of human error and helps to ensure that the resulting embryo is transferred to the correct patient. Automated witnessing systems can also be incorporated into the incubator imaging system, recording bar codes of dishes while monitoring embryo development. The data can be transmitted to a computer for continuous monitoring either on site or remotely.
Electronic witnessing systems have the advantage that they can be programmed to include traceability for all the media and batches of consumables used for a specific case. They also provide information on who performs specific procedures and the time that it takes for completion, which is very useful for audit purposes. However, the systems can also breed a new generation of errors: it is essential that full risk assessments to evaluate operation in a specific laboratory and validation checks are performed before introducing e-witnessing, rather than just accepting that an electronic system will work efficiently.
Ambient Air Quality
The quality of ambient air is a factor that may impact successful embryo development; strict management of the laboratory environment to eliminate potential contamination can contribute toward optimizing IVF outcomes (see Morbeck, 2015 and Elder et al., 2015 for reviews).
The specific air quality requirements for IVF laboratories in terms of airborne particulates vary by country/region. Air cleanliness is classified according to the number and size of particles within a sample of air, measured in particles per cubic foot or cubic meter of air.
Laboratory air quality should be maintained by use of positive air pressure relative to adjacent areas; the airflow pressures in clean areas should be continuously monitored and frequently recorded. Adjacent rooms with different clean area classification should have a pressure differential of 10–15 pascals, with the highest pressure in the most critical areas. High-efficiency particulate air (HEPA) filters that remove particles smaller than 0.3 µm and filters to remove volatile organic compounds, which can adversely affect the health of human gametes and embryos, can also be used (Cohen et al., 1997; Cutting et al., 2004). Filters must be regularly inspected and changed to ensure that their efficiency is not reduced.
The environment in a clean room is produced by incorporating parallel streams of HEPA-filtered air (laminar flow) that blow across the room to expel any dust and particles in the airflow by the shortest route, moving at a uniform velocity of 0.3–0.45 meters per second. The air velocity over critical areas must be at a sufficiently high level to sweep particles away from the area and ensure that particles do not thermally migrate from the laminar flow. At least 20 changes of air per hour are usually required for clean rooms classified as Grade B, C and D (see Table 8.1).
Table 8.1a Comparison of air classification systems
| WHO GMP | US 209E | US Customary | ISO/TC (209) ISO 14644 | EEC GMP |
|---|---|---|---|---|
| Grade A | M 3.5 | Class 100 | ISO 5 | Grade A |
| Grade B | M 3.5 | Class 100 | ISO 5 | Grade B |
| Grade C | M 5.5 | Class 10 000 | ISO 7 | Grade C |
| Grade D | M 6.5 | Class 100 000 | ISO 8 | Grade D |
Table 8.1b Classification of clean areas in terms of airborne particles
| Grade | At rest | In operation | ||
|---|---|---|---|---|
| Maximum permitted number of particles/m3 | ||||
| 0.5–5.0 μm | >5 μm | 0.5–5.0 μm | >5 μm | |
| A | 3500 | 0 | 3500 | 0 |
| B | 3500 | 0 | 350 000 | 2000 |
| C | 350 000 | 2000 | 3 500 000 | 20 000 |
| D | 3 500 000 | 20 000 | Not defined | Not defined |
The airflow can either be vertical downflow (entering via filters in the roof, exiting through vents in the floor) or horizontal crossflow (entering through filters in one sidewall and exhausted above the floor in the sidewall opposite and/or recirculated via a bank of filters).
The US Federal Standard 209E defines air quality based upon the maximum allowable number of particles 0.5 μm and larger per cubic foot of air: Class 1, 100, 1000, 10 000 and 100 000. The lower the number, the cleaner the air. The ISO classifications are defined as ISO Class 1, 2, 3,4, etc. through to Class 9. The cleanest, ultrapure air is Class 1. Guidelines suggested by regulatory and legislative authorities for IVF laboratories are based on those for Good Manufacturing Procedures (GMP), which define four grades of clean area (A–D) for aseptic handling and processing of products that are to be used in clinical treatment (Tables 8.1a and 8.1b). Each area has recommendations regarding required facilities, environmental and physical monitoring of viable and nonviable particles, and personnel attire.
Grade A (equivalent to Class 100 [US Federal Standard 209E], ISO 5 [ISO 14644–1]) is the most stringent, to be used for high-risk operations that require complete asepsis, carried out within laminar flow biological safety cabinets (BSC).
Grade B (equivalent to Class 100, ISO 5) provides the background environment for a Grade A zone, e.g., clean room in which the BSC is housed.
Grade C (equivalent to Class 10 000, ISO 7) is a clean area for carrying out preparatory stages in manufacture of aseptically prepared products, e.g., preparation of solutions to be filtered.
Personnel entering all grades of clean area must maintain high standards of hygiene and cleanliness at all times, and should not enter clean areas in circumstances that might introduce microbiological hazards, i.e., when ill or with open wounds. Changing and washing procedures must be defined and adhered to, with no outdoor clothing introduced into clean areas. Wearing of watches, jewelry and cosmetics is discouraged. Changing rooms for outdoor clothing should lead into a Grade D area (not B or C). Protective clothing for the different areas is defined:
Grade D: protective clothing and shoes, hair, beard, mustache covered.
Grade C: single or two-piece suit with high neck, wrists covered, shoes/overshoes, hair beard mustache covered; non-shedding materials.
Grade A and B: headgear, beard and mustache covered, masks, gloves, non-shedding materials, and clothing should retain particles shed by operators.
Grade D (equivalent to Class 100 000, ISO 8) is a clean area for carrying out less critical stages in manufacture of aseptically prepared products, such as handling of components after washing.
The Tissue and Cells Directive issued by the European Union (Directive 2003/94/EC) stipulates that where human cells and tissue are exposed to the environment during processing, the air quality should be Grade A with the background environment at least equivalent to Grade D, unless a less stringent air quality may be justified and documented as achieving the quality and safety required for the type of tissue and cells, process and human application concerned. Since there is no documented evidence of disease transmission in ART treatment that can be attributed to air quality in the laboratory, the European Society for Human Reproduction and Embryology (ESHRE) suggests that less stringent air quality is justified for ART (de los Santos et al., 2016).
The regulatory authority in the UK (Human Fertilisation and Embryology Authority, HFEA) recommends that all work in the IVF laboratory be carried out in Class II flow cabinets delivering Grade C quality air to ensure safe handling of gametes and embryos, with the background environment as close as possible to Grade D (www.hfea.gov.uk).
Cell culture CO2 incubators equipped with a HEPA filter airflow system can continuously filter the entire chamber volume every 60 seconds, providing Class 100/Grade B air quality within 5 minutes of closing the incubator door. These incubators may also incorporate a sterilization cycle (Steri-Cycle™) and can be supplied with an additional ceramic filter that excludes volatile low molecular weight organic and inorganic molecules, collectively known as volatile organic compounds (VOCs). Some air purification systems also incorporate UV filters.
Volatile Organic Compounds
The importance of ambient air and the possible consequences of chemical air contamination have been repeatedly reviewed (Morbeck, 2015; Elder et al., 2015). Whereas most organisms and species are protected to some extent from hazards in their ambient environment through their immune, digestive and epithelial systems, oocytes and embryos in vitro have no such protection, and their active and passive absorption mechanisms are largely indiscriminate. IVF laboratories set up in buildings within polluted areas, or close to airports or industrial manufacturing sites, may be subject to serious chemical air contamination, which may be reflected by inadequate pregnancy and live birth rates. Large traditional incubators obtain their ambient air directly from the laboratory room; gas mixtures are supplied in gas bottles, which may be contaminated with organic compounds or metallic contaminants. Pressurized rooms using HEPA filtration are used by many IVF laboratories, with standards applied to pharmaceutical clean rooms; however, HEPA filtration cannot effectively retain gaseous low molecular weight organic and inorganic molecules.
The four most common air pollutants are:
1. Volatile organic compounds: in urban and dense suburban areas, VOCs are produced by industry and by vehicle and heating exhausts, as well as by a variety of cleaning procedures. Instruments such as microscopes, television monitors or furniture (as a result of manufacturing processes) may also produce VOCs; perfumes, after-shave and other highly scented aerosols are also potential sources, and all theater and laboratory staff should be discouraged from their use.
2. Small inorganic molecules such as N2O, SO2 and CO.
3. Substances derived from building materials, such as aldehydes from flooring adhesives, substituted benzenes, phenol and n-decane released from vinyl floor tiles; flooring adhesives have been found to be particularly aggressive in arresting embryo development. Newly painted surfaces frequently present a hazard, as many paints contain substances that are highly toxic in the IVF laboratory; laboratory renovations and painting should always be planned during a period when treatment cycles are not being performed.
4. Other polluting compounds which may be released by pesticides or by aerosols containing butane or iso-butane as a propellant. Liquids such as floor waxes may contain heavy metals, which have a drastic effect on embryo implantation potential.
Detailed studies of chemical air contamination in all areas of an IVF laboratory have revealed that there are dynamic interactive processes between air-handling systems, spaces, tools, disposable materials and other items unique to the laboratory. Anesthetic gases, refrigerants, cleaning agents, hydrocarbons and aromatic compounds may be detected, and some of these can accumulate specifically in incubators. Water-soluble and lipid-soluble solid phases such as those in incubators can interact: whereas some contaminants may be absorbed by culture media, this may be counteracted by providing a larger sink such as a humidification pan in the incubator. Mineral oil may act as a sink for other components. Unfiltered outside air may be cleaner than HEPA-filtered laboratory air or air obtained from incubators, due to accumulation of VOCs derived from adjacent spaces or specific laboratory products, including sterile Petri dishes. Standards for supplies of compressed gases are based upon criteria that are not designed for cultured and unprotected cells, with no perspective of the specific clean air needs of IVF. New incubators can have VOC concentrations more than 100-fold higher than used incubators from the same manufacturer; allowing the emission of gases from new laboratory products is crucial. Systems that can clean the air and reduce VOCs can be installed into existing air conditioning systems.
VOCs can be measured in the laboratory using sensitive hand-held monitors that use photoionization monitors to screen equipment and consumables and pinpoint sources of VOCs. Active filtration units with activated carbon filters and oxidizing material can be placed inside cell culture incubators or in the laboratory spaces themselves (Cohen et al., 1997; Boone et al., 1999). As always, prevention is the best strategy, and efforts should be made to eliminate potential sources such as alcohol disinfectants and anesthetic gases – as well as perfumes/after-shave lotions – from the laboratory.
Biological Safety Cabinets
A biological safety cabinet or BSC is an enclosed workspace that provides protection either to workers, the products being handled or both. BSCs provide protection from infectious disease agents, by sterilizing the air that is exposed to these agents. The air may be sterilized by UV light, heat or passage through a HEPA filter that removes particles larger than 0.3 µm in diameter. BSCs are designated by class, based on the degree of hazard containment and the type of protection they provide. In order to ensure maximum effectiveness, certain specifications must be met:
(a) Whenever possible, a 30-cm clearance should be provided behind and on each side of the cabinet, to ensure effective air return to the laboratory. This also allows easy access for maintenance.
(b) The cabinet should have 30- to 35-cm clearance above it, for exhaust filter changes.
(c) The operational integrity of a new BSC should be validated by certification before it is put into service, or after it has been repaired or relocated.
(d) All containers and equipment should be surface decontaminated and removed from the cabinet when the work is completed. The work surface, cabinet sides and back, and interior of the glass should be wiped down at the end of each day. (70% ethanol and FertisafeTM are effective disinfectants).
(e) The cabinet should be allowed to run for 5 minutes after materials are brought in or removed.
BSC Classes
Class I (Figure 8.1a) – provides personnel and environmental protection, but no product protection. Cabinets have an open front, negative pressure and are ventilated. Nonsterile room air enters and circulates through the cabinet. The environment is protected by filtering exhaust air through a 0.3-µm HEPA filter. The inward airflow protects personnel as long as a minimum velocity of 75 linear feet per minute (lfpm) is maintained through the front opening. This type of cabinet is useful to enclose equipment or procedures that have a potential to generate aerosols (centrifuges, homogenizing tissues, cage dumping), and can be used for work involving microbiological agents of moderate to high risk.
Figure 8.1 (a, b, c) Schematic diagrams of airflow in biological safety cabinets.
Class II – incorporates both charcoal and HEPA filters to ensure an environment that is close to sterile. It provides product, personnel and environment protection, using a stream of unidirectional air moving at a steady velocity along parallel lines (‘laminar flow’). The laminar flow, together with HEPA filtration, captures and removes airborne contaminants and provides a particulate-free work environment. Airflow is drawn around the operator into the front grille of the cabinet, providing personnel protection. A downward flow of HEPA-filtered air minimizes the chance of cross-contamination along the work surface. Exhaust air is HEPA filtered to protect the environment, and may be recirculated back into the laboratory (Type A, Figure 8.1b) or ducted out of the building (Type B).
Class II cabinets provide a microbe-free environment for cell culture and are recommended for manipulations in an IVF laboratory. They can be modified to accommodate microscopes, centrifuges or other equipment, but the modification should be tested and certified to ensure that the basic systems operate properly after modification. No material should be placed on front or rear grille openings, and laboratory doors should be kept closed during use to ensure adequate airflow within the cabinet. The laminar flow of air can have a significant cooling effect on culture dishes; some laboratories choose to switch off the flow of air at appropriate times when it is safe to do so.
Class III – is used for routine anaerobe work and is designed for work with high-risk organisms in maximum containment facilities. This cabinet provides maximum protection to the environment and the worker. It is completely enclosed with negative pressure, plus access for passage of materials through a dunk tank or double-door pass through box that can be decontaminated between uses. Air coming into and going out of the cabinet is HEPA filtered, and exhaust air passes through two HEPA filters or a HEPA filter and an air incinerator before discharge to the outdoors. Infectious material within the cabinet is handled with rubber gloves that are attached and sealed to ports in the cabinet (Figure 8.1c).
Horizontal laminar flow ‘clean bench’ – provides only product protection, and is not a BSC. HEPA-filtered air is discharged across the work surface toward the user. These can be used for clean activities but should never be used when handling cell cultures or infectious materials.
Vertical laminar flow ‘clean bench’ – is also not a BSC, but is useful in hospital pharmacies for preparation of intravenous drugs. Although they generally have a sash, the air is usually discharged into the room under the sash.
Water Quality
Although the majority of media required for an IVF laboratory are now commercially available, if any solutions are to be prepared ‘in house,’ a reliable source of ultrapure water is a critical factor. A pure water source is also required for washing and rinsing nondisposable equipment. Weimer et al., (1998a) carried out a complete analysis of impurities that can be found in water: this universal solvent provides a medium for most biological and chemical reactions, and is more susceptible to contamination by other substances than any other common solvent. Both surface and ground water are contaminated with a wide range of substances, including fertilizers, pesticides, herbicides, detergents, industrial waste effluent and waste solvents, with seasonal fluctuations in temperature and precipitation affecting the levels of contamination. Four categories of contaminants are present: inorganics (dissolved cationic and anionic species), organics, particles and microorganisms such as bacteria, algae, mold and fungi. Chlorine, chloramines, polyionic substrates, ozone and fluorine may be added to water during treatment processes, and must be removed from water for cell culture media preparation.
In water purification, analysis of the feed water source is crucial to determine the proper filtration steps required, and water-processing protocols should be adapted to meet regional requirements. Processing systems include particulate filtration, activated carbon cartridge filtration, reverse osmosis (RO) and electrodeionization (EDI), an ultraviolet oxidation system, followed by a Milli-Q PF Plus purification before final filtration through a 0.22-mm filter to scavenge any trace particles and prevent reverse bacterial contamination from the environment.
IVF laboratory personnel should be familiar with any subtle variations in their water source, as well as the capabilities of their water purification system, and develop protocols to ensure consistently high-quality ultrapure water supplies, following manufacturers’ instructions for monitoring, cleaning, filter replacement and maintenance schedules.
Supplies
A basic list of supplies is outlined in the Appendix at the end of this chapter; the exact combination required will depend upon the tissue culture system and techniques of manipulation used. Disposable supplies are used whenever possible and must be guaranteed nontoxic tissue culture grade, in particular the culture vessels, needles, collecting system and catheters for oocyte aspiration and embryo transfer. Disposable glass pipettes for gamete and embryo manipulations can be purchased presterilized and packaged. If nonsterile disposable pipettes are purchased, they must be soaked and rinsed with tissue culture grade sterile water and dry heat sterilized before use. In preparing to handle gametes or embryos, examine each pipette and rinse with sterile medium to ensure that it is clean and residue-free.
Important considerations in the selection of supplies include:
Suitability for intended purpose
Suitable storage facilities (i.e., storerooms away from excessive heat/direct sunlight)
Compliance of suppliers to a contract with specified terms and conditions
Disposable plastics CE marked or mouse embryo tested where possible
Media with quality certification, mouse embryo tested and proven track record, with validation evidence
Delivery of perishable items such as media under controlled conditions
Batch numbers to be recorded for quality assurance purposes
Health and safety of operators handling potentially infectious bodily fluids.
Routine schedules of cleaning, maintenance and servicing must be established for each item of equipment, and checklist records maintained for daily, weekly, monthly and annual schedules of cleaning and maintenance of all items used, together with checks for restocking and expiry dates of supplies.
Tissue Culture Media
Original IVF culture systems were based on simple media developed for organ explant and somatic cell culture, designed to mimic physiological conditions. Analysis of tubal and uterine fluids, together with research into embryo metabolism, then led to the development of complex media. Many controlled studies have shown fertilization and cleavage to be satisfactory in a variety of simple and complex media (see Poole et al., 2012; Mantikou et al., 2013; Swain et al., 2016 for reviews). However, the culture medium is only one component of a culture system; its efficacy is dependent on numerous other parameters, making it difficult/impossible to assess and compare the contribution of different media formulations to embryo viability. Poole et al. (2012) identified a total of over 200 factors that may affect the outcome of an IVF procedure, of which culture media represents only one of >100 variables associated with the laboratory/culture system itself; many of these parameters can also have a direct impact on the efficacy of the culture media itself. Specific culture parameters and their effects on embryonic development have been further reviewed and discussed by Wale & Gardner (2016).
Metabolic and nutritional requirements of mammalian embryos are complex, stage specific and, in many cases, species specific; several decades of research in laboratory and livestock animal systems have shown that, although there are some basic similarities, culture requirements of different species must be considered independently. Understanding metabolic pathways of embryos and their substrate and nutrient preferences has led to major advances in the ability to support embryo development in vitro; the physiological role of the oviduct in vivo, and the composition/role of oviductal fluid should not be neglected (Ménézo et al., 2013, 2014).
Osmolarity = osmoles of solute particles per liter of solution
Temperature-dependent (volume changes with temperature)
Osmolarity of tubal/uterine secretions = 275–305 mOsm/L
Osmolality = the osmotic concentration of a solution, mOsm/kg of solvent
Temperature-independent
One osmole = Avogadro’s number of osmotically active particles = 6.02 × 1023
Physiological pH range of body fluids = 7.2–7.5. pH is related to an equilibrium between gas phase CO2 and CO2/HCO3 dissolved in the medium, with carbonic acid as an intermediate.
Henderson–Hasselbach equation:
CO2gas↔CO2dissolved↔H2CO3+H++HCO3pH=pKa+log10H+=logof the reciprocal of the molarconcentration of hydrogen ionspKa=ionization constantforthe acid
pH is affected by:
Temperature (less CO2 dissolved at higher temperature)
Atmospheric pressure (more CO2 in solution at higher pressure)
Presence of other solutes such as amino acids and complex salt mixtures, since pKa is a function of salt concentration.
HEPES buffered medium has been equilibrated to a pH of 7.4 in the presence of bicarbonate; exposure to a CO2 atmosphere will lower the pH, and therefore culture dishes containing HEPES buffered medium should be equilibrated at 37°C only, and not in a CO2 incubator.
Temperature fluctuations during storage and handling can affect pH, and should therefore be avoided:
acid pH destroys glutamine and pyruvate
some salts or amino acids may precipitate out of solution and further affect pH.
Fertilization can be achieved in very simple media such as Earle’s, or a TALP-based formulation, but the situation becomes more complex thereafter. (See Chapter 5 for details of embryo metabolism.) Prior to 1997, single media formulations were used for all stages of IVF. However, research in animal systems during the 1990s led to elucidation of the metabolic biochemistry and molecular mechanisms involved in gamete maturation, activation, fertilization, genomic activation, cleavage, compaction and blastocyst formation. This drew attention to the fact that nutrient and ionic requirements differ during all these different stages. Inappropriate culture conditions expose embryos to cellular stress which could result in retarded cleavage, cleavage arrest, cytoplasmic blebbing, impaired energy production, inadequate genome activation and transcription. Blastocyst formation is followed by an exponential increase in protein synthesis, with neosynthesis of glycoproteins, histones and new surface antigens.
Although the specific needs of embryos during their preimplantation development have by no means been completely defined, both single-step and sequential, stage-specific and chemically defined media are used in IVF systems. Media formulations endeavor to mimic the natural in-vivo situation and take into account the expected theoretical changes in embryo physiology and metabolism that occur during the preimplantation period, although this has largely been based upon mouse embryo culture (see Fleming et al., 1987; Elder et al., 2015; Ménézo et al., 2018). Protocols that have been used include:
1. One-step culture using a single medium formulation (nonrenewal monoculture)
2. Single medium formulation, renewed on Day 2/Day 3 (renewal monoculture)
3. Two-step culture using two different media formulations (sequential media culture).
Although two-step sequential media appeared to have advantages, doubts have been raised as to whether these more complex protocols have any advantage over one-step protocols. (See Biggers and Summers, 2008 for review.) The introduction of time-lapse technology led to the development of new single-step formulations, but it is impossible to assess whether any improvements in outcome might be due to the media itself, or to other factors inherent in the system, such as new incubator design or lowered cellular stress due to less handling for routine observation.
Commercial media is ‘ready to use,’ with protein supplements added, and may also contain other components or factors. Different formulations are available for sperm preparation, oocyte washing during retrieval, insemination/fertilization, early cleavage, blastocyst development and freezing/thawing. Media containing HEPES, which maintains a relatively stable pH even in ambient air, can be used for sperm preparation, oocyte harvesting and washing, and during ICSI procedures; however, HEPES-buffered medium will become acidic when placed in a CO2 atmosphere, and therefore the gametes should be washed in HEPES-free medium before being placed in the culture incubator.
