Welcome to chapter 3.

The following chapter is called "Setting up an ART Laboratory".

The author is Joyce Mathew.

The primary function of an ART laboratory is to provide an optimal environment

for gametes and embryos. To set up an ART laboratory, that is efficient and
safe, the three key areas to focus on would be the place or location of the
laboratory, the embryologists, and the protocols or procedures.
The following chapter will focus on the physical layout of the lab, the basic
equipment required and consumables used in the ART lab. The responsibilities
of the key people in the laboratory, the embryologists and the importance of
the procedures and protocols will be elucidated.
Some of the suggestions are based on the guidelines provided by the Human
Fertilization and Embryology Authority (HFEA) code of practice, 7th edition,
and American Society for Reproductive Medicine (ASRM).

First and foremost choosing the location is very crucial when setting up a
laboratory with high standards. The IVF laboratory should provide a safe, non
toxic, stable, and a pathogen free environment. Therefore, careful
consideration should be given when choosing the location.
Laboratory conditions are of paramount importance in maintaining consistent
success rates. A major area of concern is the negative impact of poor air
quality on embryonic development (Cohen 1997). Ideally the laboratory should
be away from polluted areas such as industries or areas where there is high
volume of traffic. Locations adjacent to parking lots, gasoline service stations
and construction areas should also be avoided to limit the adverse impacts of
pollutants to cell tissue culture (Hall 1998).
Cohen J, Gilligan A, Esposito W, Schimmel T, and Dale B Ambient air and its
potential effects on conception in vitro. Hum.Reprod. 1997; 12: 1742-1749
Hall, J, Gilligan A, Schimmel T, Cecchi M, Cohen J, The origin, effects and
control of air pollution in laboratories used for human embryo culture.
Hum.Reprod. 1998; 13(Suppl 4): 146-155.

When starting a new facility for IVF, the design of the laboratory should be
logically planned according to the projected workload anticipated. It should be
adjacent to the procedure room or operating theatre (American society for
reproductive medicine ASRM 2008).
The design of the lab is extremely crucial with regards to the structural design
and the environmental design. It has been shown that both laboratory structure
and air handling systems may affect air composition. With proper engineering
and material selection it is possible to reduce such contamination (Cohen
1998).
Environmental design is to ensure that the quality of ambient air is optimum for
all procedures. Structural design refers to the layout of the laboratory and
specifications with regards to the bench height, flooring, wall paint, and lighting
etc. This planning should be done with the help of clean room experts to try
and eliminate volatile organic compounds (VOCs) and chemical air
contaminants (CAC) in the laboratory.
The American society for reproductive medicine. Revised guidelines for human
embryology and andrology laboratories. Fertil Steril. 2008; 90:S45-59
Cohen J, Gilligan A and Willadsen S Culture and quality control of embryos.
Hum.Reprod. 1998;13 (Suppl.3): 137-144.

The ambient air in the laboratory should be highly sterile. This is achieved by a
heating, ventilation air conditioning system (HVAC), and an air handling unit
that supplies only to the ART laboratory. Pre filters should be placed on the
roof at the fresh air supply inlet. Activated carbon filters and potassium
permanganate filters should be placed next to the pre filters at the supply inlet
to absorb contaminants. These filters should be replaced regularly to maintain
the quality of air that is supplied into the laboratory.
High efficiency particulate air (HEPA) filters or Ultra low penetration air should
also be placed on the ceiling of the laboratory (Boone 1999).
There should be at least 18 air exchanges per hour. The laboratory should be
pressurized to prevent air from adjacent rooms entering the laboratory.
There should be activated carbon filters placed within the laboratory to absorb
volatile organic compounds (VOCs) emitted from electrical equipment, new
furniture, etc. (Cohen 1998).
Boone WR, Johnson JE, Locke AJ, Crane MM 4th, Price TM. Control of air
quality in an assisted reproductive technology laboratory. Fertil. Steril. 1999;
71: 150-154
Cohen J, Gilligan A and Willadsen S Culture and quality control of embryos.
Hum.Reprod. 1998;13 (Suppl.3): 137-144.

Walls of the laboratory should be painted with non odor emitting, nontoxic,
glossy, non-vaporizing paint. Ceiling should be solid. Flooring should be slip
proof preferably vinyl which is easy to clean.
Cupboards and tabletops should be laminated with matt finish that is washable
and made of heat resistant material. Furniture should be of normal working
height and should be ergonomically designed to reduce fatigue for staff.
Lighting should be adjustable by dimmer. There should be enough electrical
outlets.
All important equipment like the incubators, micromanipulator, refrigerator,
freezing machine etc. should be on uninterrupted power supply with trigger
alarm feature (Gianaroli 2000). There should be a wall mounted data logger
displaying the laboratory ambient temperature and humidity levels.
Gianaroli L, Plachot M, Van Kooij R, Al Hasani S, Dawson K, DeVos, Magli
MC, Mandelbaum J, Selva J and Van Inzen W. Guidelines for good practice in
IVF laboratories. Hum.Reprod. 2000; 15: 2241-2246

TThe layout of the laboratory must be sensibly planned and logically designed
to allow smooth running of routine procedures.
The ART laboratory is a dedicated facility for use in IVF related work only. It is
restricted to ART laboratory staff. After donning theatre gown, shoes and cap,
entry to the lab is through an air shower which has HEPA filters. The wash
area has a wash basin for scrubbing and another designated area for washing
and packing.
The main laboratory consists of designated areas for oocyte and embryo
culture and for ART sperm preparation. Adjacent to the main culture area is the
procedure room / theatre. The two areas are connected through an access
window through which follicular aspirates are passed into the laboratory.
Each workstation should be self contained with a laminar flow hood, incubator,
and microscope to prevent unnecessary movement of staff in the laboratory
during procedures.
There should also be allocation of separate space for micromanipulation
procedures, cryopreservation, data or record keeping and storage area for
storage of consumables.

Incubators are the most crucial equipment in the ART laboratory. The
incubators are maintained at 37C. Embryo culture media are buffered by
bicarbonate and therefore in order to maintain correct pH of the culture
medium, the commonly used gas phases are 5% or 6% CO2 in air or triple gas
that consists of 6% CO2, 5% O2 and 89% N2. Low oxygen tension has proven
to be the preferred choice (Meintjes 2009).
Accuracy of the temperature can be checked by using calibrated thermometer
or thermocouple. The pH of the bicarbonate buffered culture medium in the
incubator is maintained by CO2 and checked by a pH meter.
The incubators are used for pre-equilibration of culture medium, sperm
incubation, oocyte and embryo culture.
There should be enough incubators in the laboratory and incubators with 4-6
compartments are preferred. Frequent closing and opening incubator doors
should be avoided (Abramczuk and Lopata 1986) as this leads to fluctuation of
temperature, pH and osmolarity.
Meintjes M, Chantlis SJ, Douglas JD, Rodriguez AJ, Guerami AR, Bookout
DM, Barnett BD and Madden JD. A controlled randomized trial evaluating the
effect of lowered incubator oxygen tension on live births in a predominantly
blastocyst transfer program. Hum.Reprod. 2009; 24(2): 300-307
Abramczuk JW and Lopata A. Incubator performance in the clinical in vitro
fertilization program: importance of temperature conditions for the fertilization
and cleavage of human oocytes. Fertil.Steril. 1986; 46: 132-134.

Temperature control during ART procedures is extremely important when

handling oocytes and embryos. It has been shown that brief exposure of
human oocytes to room temperature results in spindle disruption and
microtubule disassembly (Pickering et al 1990).Therefore every effort should
be made to ensure that there is no temperature fluctuation during any
laboratory manipulation that involves the handling of oocytes or embryos and
that the temperature is maintained at 37 C.
Laminar flow hoods together with HEPA filtration, captures and removes
airborne contaminants and provides a particulate free work environment. The
stereo microscope can be built into the laminar flow cabinet and the entire
table top can be heated to maintain temperatures at 37C. This should be used
for handling oocytes and embryos.
All microscopes should have a heated stage maintained at 37C. Inverted
microscopes which have a larger working distance are used during
micromanipulation and embryo monitoring. Upright microscopes are used for
sperm evaluation. The microscope should be attached to a video camera
system for teaching and assessment purposes.
Pickering SJ, Braude PR, Johnson MH, Cant A and Currie J. Transient cooling
to room temperature can cause irreversible disruption of the meiotic spindle in
the human oocyte. Fertil Steril 1990; 54: 102-108

Micromanipulator systems are set up in an isolated area of the lab which is

free from other laboratory activities.
The entire system consists of an inverted microscope, micromanipulator arms,
and microinjector. Inverted microscopes used for micromanipulation should be
equipped with 4x, 10x, and 20x objectives with a heated stage and the entire
microscope should be mounted on a vibration free platform.
Two identical micromanipulator arms are mounted on both sides of the
microscope. These arms are capable of three dimensional movements. They
allow manipulation of the injection pipette on the right side and holding pipette
on the left side. The microinjector consists of a syringe and silicone tubing
connected to the microtool at the end of the tubing. These microtools for
holding the oocyte and for sperm injection are commercially available.
These systems are used for procedures like intracytoplasmic sperm injection
(ICSI), embryo biopsy etc.

Centrifuge is required to prepare sperm for IVF insemination or

intracytoplasmic spermatozoa injection (ICSI). Centrifuge needs to be regularly
cleaned and maintained.
A medical refrigerator is crucial for storage of culture medium. The
temperature of the refrigerator and freezer is normally charted on the
temperature chart.
The water purification system is a source of ultrapure water which is essential
in an ART laboratory and for media preparation (Weimer et al 1998). Ultrapure
water is most crucial in preparation of glassware (particularly glass pasteur
pipettes) that are used for handling gametes and embryos. The glass pipettes
are soaked in ultrapure water before they are washed, packed, and sterilized.
These systems are very expensive and require regular maintenance and
regular replacement of various filters.
Wiemer KE, Anderson A and Stewart B. The Importance of water quality for
media preparation. Hum. Reprod. 1998; 13 (Suppl. 4): 166-172

In ART, nowadays disposable consumables are used. Dry heat sterilization

oven is used when preparing glass pasteur pipettes. The pipettes after soaking
in ultra pure water are washed and packed and then sterilized in the dry heat
oven. Metal blocks, stainless steel test tube racks and silicon tubings are
sterilized daily in the oven at the end of the day. Ovens need to be checked
routinely for accuracy of temperature.
Test tube warmers are used for maintaining the temperature of follicular
aspirates contained in test tubes at 37C.
Suction pumps, though used in the procedure room for follicular fluid
aspiration, are the responsibility of the ART laboratory staff. They should be
serviced routinely and sent for recalibration to ensure that the suction levels
are accurate.

Slow freezing is achieved by the freezing machine where there is a very

gradual drop in temperature. The whole freezing program takes about 2 to 2
hours. This method of freezing can be used for cleavage stage embryos,
blastocysts and ovarian tissue. These machines are very expensive and need
to be serviced regularly. However, with recent breakthroughs in vitrification,
freezing protocols requiring these sophisticated machines may be replaced by
vitrification protocols (Balaban et al 2008).
Liquid nitrogen tanks are used for storage of cryopreserved gametes, embryos
and ovarian tissue. The specimen can be stored either in the liquid phase or
vapor phase. These tanks are topped up regularly with liquid nitrogen and are
fitted with probes that trigger alarm if low level of liquid nitrogen levels is
detected.
Balaban B, Urman B, Ata B, Isiklar A, Larman MG, Hamilton R and Gardner
DK. A randomized controlled study of human Day3 embryo cryopreservation
by slow freezing or vitrification: vitrification is associated with higher survival,
metabolism and blastocyst formation. Hum.Reprod. 2008; 23(9): 1976-1982.

Purchasing of equipment in the new setup is very crucial. Proven or

established models that have been used successfully in other successful
centers should be bought. Equipment of the highest quality and reliability
should be purchased. Equipment should be safe to use and electrical leakage
tests should be performed by biomedical engineers before commissioning the
equipment.
Regular servicing and maintenance of equipment is mandatory. For this
reason, equipment that has maintenance support and reliable breakdown
service should be used. It is important that the suppliers have spare equipment
as backup in the event of any malfunction of equipment.
All IVF equipment should be on urgent power supply. Crucial equipment like
incubators, micromanipulator systems, medical refrigerator, freezing machines
etc should be on uninterrupted power supply with a trigger alarm or call back
service.

After the completion of the new laboratory, there are few important points to
consider. The laboratory should be cleaned and all the equipment prior to
moving into the laboratory should be wiped down.
The ventilation rate should be increased and the air pressures should be
increased to purge the air in the laboratory to get rid of any irritants or VOCs
given out from the new materials, furniture etc. Activated carbon and
potassium permanganate filter units should be placed in strategic locations to
absorb any off gassing and irritants given out from newly constructed
materials.
New clean-room facilities such as ART laboratories undergo a lengthy curing
time (Boone et al 1999).The duration depends on the type of construction
material used, cleaning regime that is employed etc. During this burning-in
period, there should be no human traffic or activities in the laboratory.
Commissioning of the laboratory should only be done after appropriate testing
to verify that the laboratory meets the specifications for environmental and
structural design.
Boone WR, Johnson JE, Locke AJ, Crane MM 4th, Price TM. Control of air
quality in an assisted reproductive technology laboratory. Fertil. Steril. 1999;
71: 150-154

Consumables used in ART should be established brands that have been used
in successful centers and are proven to be safe for culture of human gametes
and embryos.
Plastic ware such as test tubes, petri dishes, graduated pipettes, four-well
culture dishes, embryo transfer catheters, glassware such as injection needles
and holders for ICSI, embryo transfer catheters and oocyte aspiration needles
are disposable and for single use only. Only powder free gloves should be
used. They should be of tissue culture grade and each lot of supply should
have a batch number. Expiration dates should be clearly stated.
Avoid too many different suppliers as standardization is crucial.
Sperm survival test can be used to test out new consumables or products to
check their suitability for use in the ART laboratory (Critchlow et al 1989).
Critchlow JD, Matson PL, Maureen CN, Horne G, Troup, SA, Lieberman BA.
Quality control in an in-vitro fertilization laboratory: Use of human sperm
survival studies. Hum. Reprod. 1989; 4: 545-549.

Culture medium used to be prepared in the IVF laboratories. Rigorous quality

control is essential in media preparation and it is quite laborious. Commercially
prepared high quality culture media are available which are increasingly being
used in laboratories all around the world. Again, it is important to use culture
media that have been established in the literature as suitable for culture of
human oocytes and embryos.
It is important to use media that is readily available. Only properly registered
media should be used for IVF procedures.
Each lot of medium should have a certification of pH, osmolarity, endotoxin
level testing, mouse embryo assay (MEA) (Jin et al 1998), and sperm survival
test. Each batch of media should have batch numbers, and date of production
and expiry dates should be clearly written on the product container.
If medium is manufactured in another country, it should be packaged properly
to maintain the correct storage temperature of the culture medium.
It is important that media from different companies should not be mixed. It is
best to follow the manufacturers instruction.
Jin M, Jin Z, Hyllner SJ, Svalander P. Sensitivity evaluation of a mouse embry
assay (MEA) used for qual;ity control of IVF culture media. Hum.Reprod. 1998;
13: 282

There are two types of commonly used embryo culture systems. Microdroplet
culture (Lane and Gardner 1992) with oil overlay and the other form of culture
is using normal volume of culture medium without oil overlay.
Microdroplet embryo culture with oil overlay needs to be prepared fast as the
droplet of culture medium is 10-20l. The advantage is that it prevents the
evaporation of media, thereby reducing the harmful effects of increases in
osmolarity. It also reduces changes in pH caused by a loss of CO2 from
medium when culture dishes are removed from the incubator for embryo
assessment.
Culture of embryos in four-well dishes without oil overlay is another method of
embryo culture. To reduce changes in pH caused by CO2 from medium, these
dishes can be placed in humidicribs that have an atmosphere of 5% or 6%
CO2 during embryo assessment.
Lane M and Gardner DK. Effect of incubation volume and embryo density on
the development and viability of mouse embryos in vitro. Hum.Reprod.1992; 7:
558-562

When setting up a quality IVF laboratory, the most important asset to consider
would be the embryologist. The laboratory should be directed by a qualified,
experienced, and responsible person with expertise in the field of embryology
(Gianaroli et al 2000).
The embryologists should have a good background in reproductive biology, or
biological sciences. They should have good aseptic technique. An
embryologist should be able to counsel patients and maintain close links with
the medical staff. The most important is hands-on experience in all facets of
clinical embryology which is an absolute requirement when starting a new
setup.
Other traits to look for when hiring embryologists would be honesty,
perfectionism, team worker etc.
It is the responsibility of the embryologists to ensure that environment and
processes in the laboratory are stable, non toxic and pathogen free. There
should be maintenance of optimum parameters for gametes and embryos.
Besides being able to perform all IVF procedures, an embryologist is
responsible for routine checks on equipment, consumables and environmental
conditions.
Gianaroli L, Plachot M, Van Kooij R, Al Hasani S, Dawson K, DeVos, Magli
MC, Mandelbaum J, Selva J and Van Inzen W. Guidelines for good practice in
IVF laboratories. Hum.Reprod. 2000; 15: 2241-2246
19

All newly hired staff should undergo a formal training on all procedures using
the murine model. This training should be done simultaneously as the
embryology lab is being constructed.
The training should improve skill sets and give better understanding of
procedural and physiological bases of procedures. There should be emphasis
on the strict adherence of the standard operating procedures (SOP).
After the relevant training, all embryologists handling human embryos should
be licensed by the national regulatory board. Embryologists should be given
opportunities to increase their knowledge and competence through attending
conferences, workshops, and continuing educational programs (Alper et al
2002).
There should be adequate number of staff to do all the procedures.
Understaffing would lead to non adherence to SOPs, staff dissatisfaction, and
chaos in the lab.
Alper MM, Brinsden PR, Fischer R, Wikland M. Is your IVF programme good?
Hum Reprod. 2002; 17: 8-10

Every laboratory should have a manual detailing all the procedures done in the
laboratory. The procedures should include sperm assessment and preparation
for ICSI or IVF insemination, oocyte retrieval, oocyte culture, IVF Insemination,
ICSI, fertilization check, embryo culture, preparation of embryo culture dishes,
embryo selection, embryo transfer, cryopreservation of gametes and embryos
etc.
Laboratory protocols have to be transposed into standard operating protocols
(SOP). All activities performed within the ART laboratory need to be clearly
written with sufficient details on the techniques (Kastrop 2003).
All SOPs should be signed and dated and should be made available to all
staff. There should be strict adherence to the SOP. Non adherence to SOPs
should be dealt with seriously and should not be excused.
When doing procedures there should be at least two identifiers for patients
specimen. The name and an additional identifier such as an identity number
should be used. Batch number of all consumables and culture medium of all
patients should be documented.
Kastrop P. Quality management in the ART laboratory. Reprod. Biomed. Online
2003;7: 691-694.

Double witnessing is extremely important when performing IVF procedures. It

is a safeguard instituted in many laboratories to ensure that samples of
patients are not intermixed.
There should be at least two individuals confirming, in writing, the identity of
the specimen (Pool 1997).
Witnessing should be done when transferring sperm samples during the
various steps of sperm preparation.
During oocyte retrieval, the identity of the patient and the sample being
handled should be confirmed by double witnessing.
The identity of the embryo transfer patient and the embryos for transfer should
be double checked.
Double witnessing is also done during gamete and embryo cryopreservation
and thawing.
When witnessing, both individuals should document the various steps during
which the witnessing was done on to the patients record book.
Pool TB Practices contributing to quality performance in the embryo laboratory
and the status of laboratory regulation in the US. Hum. Reprod. 1997; 12:
2591-2593.

It is the responsibility of the laboratory to consistently provide a high standard

of service. As part of the quality control, it is imperative that there is daily
monitoring and recording of the following equipment:
Incubator temperature and CO2 levels are measured daily by using calibrated
thermocouple and CO2 analyzer respectively. pH meter probes are used to
check the pH of the equilibrating culture medium in the incubator. The level of
CO2 gas in the tanks is also noted on a daily basis.
The temperature of the heated base of the laminar flow hood and biological
safety cabinet are checked using a calibrated thermometer.
The temperature of the heated stages of microscopes should be checked and
recorded.
Medical refrigerator temperature is recorded on a daily basis (Mayer 2003) and
the data logger on the refrigerator is checked for fluctuation.
Mayer JF, Jones EL, Lacey DD, Nehchiri F, Muasher SJ, Gibbons WE,
Oehninger SC. Total quality improvement in the IVF laboratory: choosing
indicators of quality Reprod.Biomed.Online 2003; 7: 695-699.

Since the IVF is an extension of the operating theatre every effort must be
taken to maintain the sterility in the lab. Strict discipline must be exercised with
regards to hand washing and maintaining the parameters in the laboratory.
The laboratory must be cleaned daily. All surfaces should be wiped down at
the end of the day. Any spillage during the course of the procedure should be
wiped with distilled water followed by dry tissue. Alcohol should not be used for
cleaning while oocytes and embryos are being handled. Waste such as
seminal fluid, follicular fluid etc. should be removed as soon as the procedure
is completed.
There should be maintenance schedule for all the crucial equipment. It can be
in the form of a service contract. Incubators which are the most important
equipment in the laboratory should be serviced twice a year. All other
equipment such as microscopes, laminar flow cabinets, medical refrigerators,
hot air sterilizing oven, centrifuge and oocyte suction pump should be done
once a year. Temperature thermocouple, pH meter and CO2 analyzer should
be recalibrated every year. All these checks are crucial for reproducibility of
results.

In conclusion, the ART laboratory plays a crucial role in the treatment of

infertile couples and setting up a state of the art laboratory is no easy task.
To setup a quality ART laboratory, the combination of the three key areas will
provide the main framework. The main goal of the laboratory should be to
provide a safe and secure environment while maintaining optimal parameters
for embryonic development. The embryologists selected should be
experienced and highly skilled and be able to deliver consistent results.
Quality standards are needed to ensure consistency and reproducibility of all
methods. Procedures and processes should be clearly written and available.
For a successful ART program, the ART laboratory should establish and
maintain strict quality controls.

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