Setting up ART ,IVF laboratory standards

Artificial Reproductive Technique By Dr,Mahboob Khan Phd The following chapter is called "Setting up an ART Laboratory". The author is Dr.Mahboob Khan Phd .

1

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 (VOC’s) 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 (VOC’s) 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

6

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.

7

Incubators are the most crucial equipment in the ART laboratory. The incubators are maintained at 37°C. 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 37°C. This should be used for handling oocytes and embryos. All microscopes should have a heated stage maintained at 37°C. 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 37°C. 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 ½ 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 VOC’s 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 manufacturer’s 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 10-20Kl. 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 SOP’s, 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 SOP’s should be signed and dated and should be made available to all staff. There should be strict adherence to the SOP. Non adherence to SOP’s 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.

26

27

28