The Art & Science of Assisted Reproductive Technology Sunita R Tandulwadkar
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1Setting-up an ART Laboratory
  • 1. How to Set-up an ART Laboratory?
  • Sunita R Tandulwadkar, Devika Chopra
  • 2. Quality Control in an ART Laboratory
  • Vijay Mangoli, Ranjana Mangoli
  • 3. Quality Care Management in ART Clinic
  • Madhuri Patil2

How to Set-up an ART Laboratory?1

Sunita R Tandulwadkar,
Devika Chopra
 
INTRODUCTION
The journey of a successful in vitro fertilization (IVF) program starts from the clinician’s office, goes through the assisted reproductive technology (ART) laboratory and ends in a successful delivery at the hospital. What most people fail to recognize is that the heart of a successful IVF or intracytoplasmic sperm injection (ICSI) cycle lies in the laboratory. The laboratory is the place where the semen is analyzed, oocytes are assessed after retrieval and finally, where embryos are formed and cryopreserved. A well-equipped and organized laboratory is what separates an excellent IVF fertility center from an average one.
Many aspects need to be looked into before setting-up an ART laboratory. The location, construction, the equipment and ventilation are a few important parameters that need to be kept in mind while implementing a successful ART program. This chapter has highlighted the important steps that need to be taken whilst starting an ART laboratory.
 
LOCATION OF THE ART LABORATORY
The location of the ART laboratory with respect to the ART unit as well as the local area in which the ART unit is placed may play an important role in the success rates of that particular ART unit. The locality of the ART unit should be in a low-traffic, secure area, which is easily accessible.1 It should be reviewed whether the building site or nearby area is scheduled to undergo construction or renovation. This is of particular importance as activity related to any type of demolition; construction or renovation will adversely affect laboratory results. Air in urban areas may contain high levels of pollutants such as carbon monoxide, nitrous oxide, sulfur dioxide and heavy metals. This may affect the ambient atmosphere within the laboratory. The importance of air handling units is discussed elsewhere in this chapter.
The laboratory should be physically isolated. It should be in proximity to the procedure room where oocyte retrieval, embryo transfer and microsurgical epididymal sperm aspiration (TESA)/percutaneous epididymal sperm aspiration (PESA) is performed. The IVF unit should be a no-smoking zone. The use of cosmetics and strong fragrances by laboratory staff should be monitored and controlled.
 
CONSTRUCTION MATERIALS
Construction materials including internal finishes, doors, air vents, floors and ceiling elements should be selected based on their durability and maintenance requirements. All surfaces should be made of smooth, impervious and non-shedding materials that offer no surface roughness or porosity which might allow retention of particulate matter or the development of microbiological contamination.
Paints, adhesives, glues and sealants may be implicated for releasing VOCs like alkanes, aromatics, alcohol, aldehyde and ketones, etc. Therefore, wall surfaces are covered with low-VOC water-based paint with acrylic, vinyl acrylic or acrylic latex polymers. Interior paint should not contain formaldehyde, acetaldehyde, isocyanates, reactive amines and phenols. Emission testing of paints should be done.2 Stainless steel and anodized aluminum can be used in doors as well as in workstations. Water-based low-VOC adhesives can be used when needed.
Indoors; construction materials such as MDF, PVC flooring, paints and adhesives constitute the major source of Volatile Organic Compounds (VOCs) leading to the phenomenon called “sick building syndrome”. Other sources of indoor chemical hazards are cleaning fluids, floor waxes, cosmetics and cigarette smoke.
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CONSTRUCTION DESIGN
The embryology laboratory should have adequate space for:
  • Aseptic and optimal handling of gametes and embryos.
  • Storage areas and equipment such as incubators, centrifuges and cryo equipment—logical planning for efficiency and safety within each working area.
  • Record keeping, data entry, and related administrative functions. Computer equipment should be available for data collection compliance.
  • A general wet area in which washing of equipment, sterilization, etc. is performed should be separate from the embryology laboratory. Moreover, if fixatives are applied, a fume-hood will be required for handling of gametes and embryos.3
When designing a laboratory, recent developments in equipment and facilities should be considered. Work-benches should have a height which permits to work comfortably. Ducts and equipment must be laid out in such a way that repair work when needed can be performed outside the laboratory, without disturbing its functionality. Incubators, laminar flow units and micromanipulation work station should be placed in such a way that an embryologist should be able to finish one complete procedure without moving more than 3 meters in any direction. Air inlets and outlets should be carefully spaced for prevention of changes in local temperature. Micromanipulation work stations and laminar flow hoods should not be placed too close to air supply fixtures that it impacts on sterility or temperature.4
 
Burning In
At the end of the construction, there should be a 3-month waiting period before first occupation of the new facilities. During this period, the temperature is increased by 10–20°C and the air-handling ventilation unit should be set up to continuously bring in fresh air. After this period, testing should be carried out for particulate matter and microbes.
 
CONFIGURATION OF THE AIR-HANDLING VENTILATION AND FILTRATION SYSTEM
It has been suggested that control of VOC levels within the ambient air is critical for successful conception in vitro.5 Outside air, which is relatively VOC free, may in fact be a cleaner source than inside air, since each of the fixed and transient laboratory components used may produce gaseous emissions. Air handling systems should probably be designed with these findings in mind (Fig. 1).6
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Fig. 1: Air-handling system
There is an association between the presence of air contaminants in the IVF laboratory and impairment of embryonic development.7 Controlling air quality in an ART laboratory has shown beneficial effects regarding fertilization and embryo development.8 Therefore IVF laboratories are equipped with various filters. The outside air brought into the unit is first filtered with activated carbon, which removes various hydrocarbons, and then high-efficiency particulate absorption (HEPA) removes the particulate materials (0.3 microns).6 The purified air should be supplied to positively pressured room (0.10–0.20 inches of water) that uses around 7–15 fresh air changes per hour (FACH). A room with positive pressure has higher air volume entering than exiting the room. As a result, it is difficult for unpurified air to enter the room from the surrounding areas. However air entry cannot be prevented when doors are opened and closed.9 The system must be capable of supplying air with temperature of 30–35% at least 40% relative humidity.
 
USE OF ULTRAVIOLET LAMPS IN IN VITRO FERTILIZATION LABORATORY
Ultraviolet radiation acts as an antibacterial agent but system’s effectiveness depends on air quality. Sterilized environment can only be achieved if the room in question is hermetically sealed and completely subjected to 254 nm ultraviolet radiation. The object is to reduce the number of microorganisms to a minimum. The lamps are installed between the activated carbon filter and HEPA filters.10
Sequential ultraviolet illumination is highly effective in eliminating or reducing fungal and bacterial conta­minants. During the construction of laboratory installation 5of a sequential ultraviolet illuminator with the intention of decreasing circulating biological contaminants is advisable.11
 
LAMINAR AIR FLOW HOODS
There are two types of laminar air flow (LAF) hoods (Fig. 2), vertical and horizontal flow. The main goal of the LAF hood is to protect the gametes and embryos. Laminar flow cabinets create particle-free working environments by projecting air through a filtration system and removing it across a work surface in a laminar or unidirectional air stream. They provide an excellent clean air environment for a number of laboratory requirements. Laminar flow cabinets work by the use of in-flow laminar air drawn through one or more HEPA filters, designed to create a particle-free working environment and provide product protection. Commonly, the filtration system comprises of a pre-filter and a HEPA filter. The laminar flow cabinet is enclosed on the sides and constant positive air pressure is maintained to prevent the intrusion of contaminated room air.12,13
 
INCUBATORS
CO2 incubator: CO2 incubators mimic the environmental conditions that sperm, oocytes, blastocysts and develop­ing embryos encounter in vivo (Figs 3A and B).
A minimum of two CO2 incubators are essential in any program as a backup in case of unexpected malfunction. One is exclusively for media equilibration and another one for fertilization/embryo culture.
Triple gas incubator: Incubators with an atmosphere comprising CO2, O2, and N2 provide a more natural environment than a plain CO2 incubator, giving better embryo quality and higher success rate. Gas cylinders should be placed outside or in a separate room with an automatic backup system.
Benchtop incubator: This system places a heated surface directly above and below the culture dish (Fig. 3C). The small volume of the chambers within this incubator allows rapid recovery from exposure to room air (lid opening). Temperature, humidity, and CO2 inside of the chamber return to pre-opening levels rapidly.
 
MICROSCOPES
Oocyte pick-up, embryo handling and micromanipulation require various types of microscopes. Most of the available models of stereo zoom microscopes, transmitted light sources and inverted microscopes can be integrated into IVF workstations.
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Fig. 2: Laminar air flow hood
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Fig. 3A: CO2 incubator
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Fig. 3B: CO2 working incubator
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Fig. 3C: CO2 benchtop incubator
Different types of microscopes used for various IVF applications:
  • Upright microscope used for sperm vitality tests and oocyte collections (Fig. 4A).
  • Inverted microscope used with micromanipulator for processes such as ICSI, intracytoplasmic morpho­logically-selected sperm injection (IMSI) (Fig. 4B). Inverted microscopes are available from Nikon, Olympus, Leica, Carl Zeiss, etc. The micromanipulators those are available in the market are Narishige, Eppendorf, Research Instruments, Cell Robotics, etc., to mention a few.
  • Stereo zoom microscopes: Stereo zoom microscope is used in IVF laboratory for egg harvesting during ovum pick up, for insemination, embryo changes from media to media, embryo loading to embryo transfer catheter, etc. Stereo zoom microscope is usually integrated into the heated laminar flow table (Fig. 4C).
 
CENTRIFUGES
Centrifuge machine with swing-out rotor is ideal for the sperm preparation in IVF (Fig. 5). Centrifugation or vigorous mixing of open containers has a high potential for creating aerosols or droplets. Centrifuges may be placed in exhaust hoods during use or non-aerosol centrifuges may be used. Capped tubes must be used for centrifugation.1
 
LIQUID NITROGEN CANS
Liquid nitrogen dewars are essential for storage of sperm and embryos (Fig. 6). Nitrogen tanks should be cleaned and sanitized at least every year. Sufficient number of dewars should be available depending on case load.
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Fig. 4A: Upright microscope
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Fig. 4B: Inverted microscope
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Fig. 4C: Stereo zoom microscope
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Fig. 5: Centrifuge
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Fig. 6: Liquid nitrogen cans
The samples must be labeled clearly before placing them into the liquid nitrogen tanks. Records must be kept so that samples are easier to find. Liquid nitrogen tanks come attached with alarms that go off when the temperature in the tank reaches a critical level that is deleterious to the stored embryos and/or gametes.
 
CONCLUSION
Many factors need to be kept in mind before setting up an ART unit and laboratory. The success of any ART program depends upon the laboratory, its equipment and its upkeep. In conclusion, IVF success rates can be correlated with a well-functioning and state-of-the-art laboratory.
REFERENCES
  1. The Practice Committee of the American Society for Reproductive Medicine and the Practice Committee of the Society for Assisted Reproductive Technology. Revised guidelines for human embryology and andrology laboratories. Fertil Steril. 2008;90(Suppl 3):S45-59.
  1. Giligan A. Guidelines for material use in USA during construction of tissue culture laboratory. New Jersey: Alpha Environmental;  2006.
  1. Gianaroli L, Plachot M, van Kooij R, Al-Hasani S, et al. ESHRE guidelines for good practice in IVF laboratories. Committee of the Special Interest Group on Embryology of the European Society of Human Reproduction and Embryology. Hum Reprod.2000;15:2241–6.
  1. Cohen J, Alikani M, Gilligan A, et al. Setting up an ART laboratory. In: Gardner DK, Weissman A, Howles CM, Shoham Z (Eds). Textbook of Assisted Reproductive Techniques, 4th edition. United Kingdom: Informa;  2011.
  1. Mahran AM, Sharma RK, Abdel-Maguid E, et al. Evaluation of sperm chromatin damage with two routine sperm processing procedures used for assisted reproduction. Fertil Steril. 2008;76(Suppl 3):S16.
  1. Cohen J, Gilligan A, Schimmel E, et al. Ambient air and its potential effects on conception in vitro. Hum Reprod. 1997;12(Suppl 8):1742–9.
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  1. Boone W, Johnson J, Locke A, et al. Control of air quality in an assisted reproductive technology laboratory. Fertil Steril. 1997;71(Suppl 1):150–4.
  1. Angtrakool P. International Standard (ISO 14644). Cleanrooms and associated controlled environments. USA: Food and Drug Administration;  2001.
  1. Mukherjee T, Duke M, Alan C, et al. Value of sequential ultraviolet illumination in reducing ambient fungi, bacteria, and nonviable fungal structures (NVFS) in an IVF laboratory. Fertil Steril. 2003;80(Suppl 3):289–90.
  1. Khoudja YF. Better IVF outcomes following improvements in laboratory air quality. J Assist Reprod Genet. 2013;30:69–76.
  1. Lee M, Grazi R, Seifer D. Incorporation of the Cook K-Minc incubator and media system into the IVF lab: the future of IVF. J Clin Embryol. 2010;13(3):21–32.
  1. Esteves SC, Bento FC. Implementation of air quality control in reproductive laboratories in full compliance with the Brazilian Cells and Germinative Tissue Directive. Reproduct Bio Medicine Online. 2013;26:9–21.