Setting up of an Assisted Reproductive Technology CenterCHAPTER 1

An assisted reproductive technology (ART) center has specific requirements which are essential for the success of any in vitro fertilization (IVF) program. There are many factors that go into making a good IVF laboratory from a construction and facility standpoint, in addition to the people who make it all work. Much depends on air quality, laboratory design and an ongoing quality control. An IVF program is only as good as the laboratory, which supports it.

Besides design of immediate culture spaces, it is important to give thought to adjacent areas as it is not possible for air conditioning units to prevent pollution inside the laboratory from the surroundings with current air-conditioning technologies. The laboratory should be away from traffic, dust and pollution, preferably on a higher floor (not ground floor). Moisture-prone areas like basements must be avoided. It should be away from the hospital traffic. High levels of carbon monoxide, nitrous oxide, sulfur dioxide and heavy metals are found in urban areas especially near busy roads, car parks and industries.

Bulky instruments need to be moved in and out while setting the laboratory and for maintenance. Hence, access to the laboratory needs to be large enough to accommodate them. Entry to laboratory should be restricted.

Power points should be made at a regular distances over working benches. They should be provided in a number over and above the estimated requirement. Supply from different electric phases and provision for uninterrupted power supply is important as there should be no interruption in power supply to the incubator and to other essential services in the clinic. It is imperative that a power back up in the form of UPS systems and/or a captive power generation system is available.

The IVF laboratory has to have a separate air conditioning unit, which is not common with the rest of the hospital air. This system should be attached to an air purifying unit.

Scrubbing and washing area will need adequate water supply and well concealed drainage.

Factors like air quality, temperature, and light are known to affect oocytes and embryos.

For a successful IVF program it is important that the laboratory air is clean. Embryos cannot fight against air pollutants as they have no immunity and pollutants can dissolve in aqueous solutions of embryo culture medium. Improved fertilization rates and embryo development were seen with superior air quality in an ART laboratory.¹ Increased NO₂ is consistently associated with lower live birth rates.² Volatile organic compounds (VOC) are important air contaminants. VOCs are isopropanol (aka Isopropyl Alcohol) benzene, hexane, formaldehyde, and vinyl chloride. Many others are hydrocarbon based compounds that are found in fuel, solvents and adhesives are released by them. VOCs may also be produced by specific instruments such as microscopes, television monitors or furniture and in substances derived from building materials, flooring, etc. Any new construction is going to release VOCs from the paint and construction materials. Also, all furniture made today is going to have some amount of particle board, which is 10% formaldehyde resin by weight, which off-gasses for over 20 years.

Comfortable and constant temperature should be maintained by air conditioning system. The embryo grows in the body at a temperature of 37°C. Decreasing the temperature only 1°C will disrupt the spindle apparatus that helps the chromosomes separate. Operating rooms temperature is raised for the retrieval and transfer techniques. Ambient temperature should be 24–26° C.

Lighting should be within sealed units. Sunlight and cool-white fluorescent office light, which is blue-white in appearance, were the most detrimental to the mice embryo development while warm-white light, which is typically used to illuminate homes and residential environments having a yellow-white color, was significantly less damaging. At 200-lux lighting, red (620–750 nm) ray yielded the best development, whereas blue (445–500 nm) decreased blastocyst formation.⁴ When exposed to light, the embryos produced increased levels of radical oxygen, which is toxic to cellular development. UV light may be a potential stress or for the embryo and is definitely unnatural. Success rate may improve if harsh lighting is replaced with warm-white light bulbs for illumination. Attempt should be made to minimize light exposure as much as possible during each stage of embryo development. Filters are used in the microscopes and rheostats are used to control the amount of light in the room when the embryos are out of the incubators.

The floors should be scratch proof and non-slippery. The edges and corners should be curved for easy cleaning. The flooring can be a single sheet that wraps up the wall for 6 inches. This keeps the chance of dust or germs in the crevices between flooring tiles low and makes it easier to clean the flooring. Tiles should be as large as possible so that there are minimum joints which may accumulate dust particles. Partition should be of non-porous inert material. False ceilings 6are not recommended as they are inaccessible for maintenance. However, they may be needed to conceal lights and filters. In that case the walls should go all the way up to the roof above the false ceiling so that laboratory is isolated. Additionally, the false ceiling should have solid panels. Walls of IVF laboratory and OT should be dark, preferably black granite with minimum joints, smooth and non-porous. Water based paint formulated with low VOC potential should be used. No paints containing formaldehyde, acetaldehyde, isocyanates, reactive amines, phenols and other water soluble volatile organics should be used. Solvent free adhesives or vinyl glue used for floor covering with low VOC emissions are available.

Adequate steps should be taken to make the whole clinic vermin proof, with suitable traps for preventing insects and other forms of unwanted creatures entering the clinic. This essential detail should be planned at an early stage because no pesticide can be used in a fully functional IVF clinic, as it could be toxic to the gametes and embryos.

The entry should be restricted and preferably be with electric eye doors. The doors should be coated or of steel, which can be cleaned with ethyl alcohol. Sliding doors should be avoided. Sealed doors and pass through windows are preferred. In order to control the laboratory environment, it is best not to have window or other surface that may not insulate the laboratory well.

Stainless steel furniture is preferable as board releases VOCs and is less easy to clean. Chairs and stools with adjustable height add to the comfort of the scientist and reduce fatigue. The working benches should be of correct height. Easily slidable drawers with adequate capacity for storage should be installed in the laboratory with working benches. The surfaces should be non-porous and easily cleanable. It is preferable not to put shelves on the wall as dust can collect on top and can go unnoticed.

An IVF clinic would require an area of 2,500 sq. feet. This would also take into consideration adding on instruments for ever-expanding technology in this sector. The IVF laboratory and the OT occupy the major space. The IVF clinic can be divided into sterile and non-sterile areas⁵ (Fig. 2).

The sterile area shall house the operation theater, an adjoining embryology laboratory and a room to keep the cryocans for freezing. Entry to the sterile area must be strictly controlled by an anteroom for changing footwear, area for changing into sterile garments and a scrub-station. The sterile area must be air conditioned where fresh air filtered through an approved and appropriate filter system is circulated at an ambient temperature (22–25°C).

The infertility clinic need not have in-house facilities to perform all the procedures necessary to diagnose infertility. They can be attached to specialty laboratories nearby.

The infertility clinic must have ready access to laboratories that are able to carry out immunoassays of hormones [follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin, human chorionic gonadotropin (hCG), thyroid stimulating hormone (TSH), insulin, estradiol, progesterone, testosterone and dehydroepiandrosterone (DHEA)] and tests such as for human immunodeficiency virus (HIV) and hepatitis B. Endocrine evaluation is necessary to estimate blood estradiol in women undergoing controlled ovarian hyperstimulation, as dose of drugs to be given for induction of ovulation is sometimes determined by estradiol levels.

Another important facility in an ART clinic (or easily accessible to it) would be that of a microbiology laboratory that can carry out rapid tests for any infection and a pathology laboratory.

Genetic counseling and testing should be available to all couples known to carry a hereditary disease.

The practice of ART requires a well-orchestrated teamwork between many specialties.⁵ This includes a gynecologist and program coordinator/director, andrologist, ulrasonologist, clinical embryologist, counselor, technicians, trained OT nursing sisters, receptionist, data entry manager and secretary.

The treating doctor must be responsible for maintaining all records of diagnosis, treatment given and consent forms. It would be the gynecologist's responsibility to see that all equipment and instruments in the operation theater are properly functional and in order, and that a logbook is maintained of their use and operation. He/she should be able to coordinate the activities of the rest of the team and take care of staff, administrative matters, stock keeping, finance, maintenance of patient records, statutory requirements, and public relations. He/she should ensure that the staff are keeping up with the latest developments in their subject, by providing them with information from the literature, making available to them access to the latest journals, and encouraging them to participate in conferences and meetings and present their data. The program coordinator/director should have a postgraduate degree in an appropriate medical or biological science.

A urologist with a postgraduate degree in urology often takes on the task of treating male infertility. An andrologist may be attached part time to the unit and has knowledge about diagnosis of various types of male infertility covering psychogenic impotence, anatomical anomalies of the penis, which disable normal intercourse, endocrine factors that cause poor semen characteristics and/or impotence, infections, and causes of erectile dysfunction.

The clinical embryologist must be knowledgeable in mammalian embryology, reproductive 11endocrinology, genetics, molecular biology, biochemistry, microbiology and in vitro culture techniques with first-hand, hands-on experience of the techniques of ART. The responsibilities of the clinical embryologist would be to ensure that all the necessary equipment and disposables are present in the laboratory and are functional, to perform all the procedures pertaining to processing, handling and culturing of gametes and embryos in the laboratory and hand over the embryo to the gynecologist and to maintain records of all the procedures carried out in the laboratory.

Counselors are an important adjunct to any infertility clinic. The patients are adequately informed of what and what not to expect from the treatment offered to them. A person who has at least a degree (preferably a postgraduate degree) in Social Sciences, Psychology, Life Sciences or Medicine, and a good knowledge of the various causes of infertility and its social and gender implications, the stress associated with it and the possibilities offered by the various treatment modalities, should be considered as qualified to occupy this position.

The laboratory equipment must be adequate for laboratory work. Crucial equipment, like incubators and cryopreservation storage facilities must have appropriate monitors and alarms. Gas cylinders should be placed outside with an automatic backup system. Devices like warm stages, heating blocks for the maintenance of temperature of media, gametes, zygotes and embryos during the time that they are out of the incubators, should be in position. Protocols for maintaining incubators and other equipment should be clearly defined on how frequently they must be sterilized and cleaned. Records of maintenance on all the equipment must be documented. Quality checks of accuracy of functional parameters for devices used to maintain temperature and CO₂ should be performed using calibrated thermometers and CO₂ analyzer and/or pH meter. Besides, instruction manual for every instrument being available, written instructions should be available to all members of the staff for actions to be taken in the case of equipment failure. New equipment should be “run in” to dissipate latent VOCs produced in their manufacture. This may make the difference between a poor initial pregnancy rate or successful start.

Carbon dioxide (CO₂) incubators are required to maintain optimum culture conditions. IVF procedures demand accurate temperature and CO₂ levels, quick and convenient incubator access within a work station, and fast recovery after the door is opened. The incubator which maintains the temperature accurately and has minimal CO₂ recovery should be preferred. Incubators may have either thermal conductivity or infrared sensors. Between water and air jacketed incubator, an air jacketed one is preferred. A minimum number of two incubators are recommended. This is for both, a backup facility, and to decreases the frequency of repeatedly opening the first, because samples are distributed. CO₂ supplied to the incubator should be of medical grade. Through a two-stage regulator ideal pressure to minimize gas consumption without affecting CO₂ recovery time is maintained. There are alarm systems connected to mobile phones, which notify the embryology staff if the incubators change temperature or CO₂ concentration. Metal gas tubing should be avoided. In line filter between CO₂ cylinder and incubator eliminates all contamination from the gas. There are large measurable differences in the ability to maintain set temperature that depends on the type of incubator chosen for IVF culture.⁶ Mini incubators have shown better temperature recovery, oxygen concentration return, and embryo and blastocyst formation rate compared with the conventional incubator.⁷ Triple gas incubators are also available. The oxygen concentration may also be altered with these. Studies have shown that blastocysts cultured in a lower oxygen concentration 5% O₂ environment consistently resulted in higher rates of live birth. Hence, oxygen concentrations may influence success rates and the role of triple gas incubators may be important.⁸

The size of the hood can be according to need. Accessories such as gas ports and humidifying systems can be added to the flow hood to control the pH and osmolarity. Flushing CO₂-gas mixture maintains and correct pH. Temperature is controlled with a built-in heated area in the table plate. Microscopes are fixed. Usually there are 0.3 micron HEPA filters and 10 microns prefilters, i.e. a class 100 where 99.99% of airborne particles of 0.3 microns and above size are removable providing dust and bacteria free atmosphere. A vertical laminar flow is preferred. Turbulence patterns 12around objects in the vertical flow hood will be different from that in horizontal-flow units.⁹

Microscopes must deliver ample depth of field as well as clarity, detail, accurate color and the least possible distortion. Dependable, high-performance optics are central to the achievement of consistent, accurate results in today's widening range of microscopy applications, as are ergonomic features which make even long-duration tasks easier and less demanding to perform.

Stereo zoom microscope: Good quality optics makes oocyte identification easy and reduces the strain on the eyes. Ergonomically placed zooming and focusing of knobs are useful. Transmitted light source, which provides even illumination should be chosen.

Binocular microscope: Binocular microscope is used for andrology. The standard microscope is provided with an objective of 10X, 20X, 40X, and 100X. A trinocular microscope with CCD camera is also available and may also be used for teaching purposes.

Inverted microscope: Inverted microscope is useful for studying the pronucleus stage and embryo morphology. Micromanipulation requires inverted microscope with Hoffman modulation or Varel contrast system making sure it can be adapted with a micromanipulator and pre-implantation genetic diagnosis (PGD) techniques in the future.

The specially selected adapter, a pair of positioners, joystick micromanipulators and universal joints are combined. Three-dimensional coarse movements are performed electrically and fine movements by an oil hydraulic mechanism can be performed. This system enables exceptionally delicate manipulations.

Ovum aspiration pump is used for oocyte retrieval. It ensures constant negative pressure for aspiration of follicular fluid (Figs 10A and B).

Purification systems use a back-position Hospital Grade HEPA Filter with a 99.997% removal rating of 0.3 micron particles. There is also four different carbon types in activated charcoal granules, specially impregnated with various compounds such as phosphoric acid, potassium permanganate, activated zeolite and certain other proprietary coatings to filter out bacteria, fungal spores, dust mites, pollen, insect dust, dander and smog infiltration.

This composition is effective on a wide variety of odors and gases, including diesel fumes, foods, hospital odors, paint solvents, VOC's and criteria air contaminants (CAC)'s, acid gases such as hydrogen sulfide, sulfuric dioxide and hydrogen chlorine, formaldehyde, and other aldehydes. The UV chamber with the catalytic converter cleans air to the last molecule. The activated photo catalyst attacks the pollutants chemical bonds, converting the toxic compounds into benign constituents such as water and carbon dioxide.

Maintaining temperature of the gametes all along the procedure in the laboratory is of prime importance and for this purpose warming blocks, petri dish warmer and stage warmer are used (Figs 11 and 12).

Programmable embryo freezer is an integrated stand alone unit with no requirement of PC for programming. It gives full range of information on the large LCD screen including graphs. It has its own internal battery for portable use. It has a temperature range of 150–100°C. The freezer has a sterilizing program designed to hold the straw plate temperature at 125°C for 30 minutes. Record keeping can be done by automatic data logging of all freezer runs.

Laser system for assisted hatching and embryo biopsy is attached to the micromanipulator. The laser is digitally controlled and includes imaging software for digital image or video processing also. The exposure of oocytes and embryos to the artificial conditions of in vitro culture may have negative effects on the embryo's ability to undergo normal hatching, resulting in low rates of implantation. Artificially, disrupting zona pellucida (assisted hatching) tends to improve success rates with these embryos.^15,16 Laser is also required for embryo biopsy.

Meiotic spindles tether the chromosomes of oocytes and can be imaged non-invasively based on their birefringence, as they are illuminated with polarized light through digital, orientation independent polarized light microscope. The safety and utility of polscope imaging has been demonstrated in women undergoing intracytoplasmic sperm injection (ICSI) where it is used to bring into view the spindle before ICSI ensuring that the spindle is not harmed by the injection pipette. Spindle imaging with the polscope provides structural information closely related to the more invasive immunostaining method, and also enables study of the dynamic architecture of spindles.^17,18

Makler sperm counting chamber is designed to produce 10 micron thick smear, which avoids overlapping of sperms, and allows free movement of sperms in all directions. The applied specimen is easily evaluated in one focal plane. The cover glass is encircled with a metallic ring. The designing is such that, when instrument is loaded with the sample, the cover glass will never rise, making observation easier and uninterrupted (Fig. 13).

An automated analysis of semen is done on the screen. The estimation of seminal parameters by CASA improves the objectivity, precision and reproducibility of values measured. Novel parameters, such as sperm velocity and characteristics of track direction, become measurable. An important advantage is direct on-line documentation. By adding an image capture rate of up to 60 frames per second, you get the highest level of accuracy available today for measuring sperm velocities and motion parameters (Fig. 14).¹⁹

A centrifuge with digital speed and time indicator is needed for semen preparation.

Ultrasound is required for oocyte retrieval and embryo transfer. The ultrasound machine must have a transvaginal probe (Fig. 15).

Heat sealing machines: Heat sealing machine is used for straws and polybags sealing.

Antivibration microscope table: An antivibration table has vibration isolators with rolling diaphragm to give isolation from surface borne vibrations in all directions.

Electronic thermometer: A thermometer with surface sensor demonstrating the accuracy of heating systems or for checking the temperature on daily routine is required.

Submersible sensor: A submersible sensor is needed to check temperature of objects or liquids like media.

Fyrite kit: Fyrite kit is fast and accurate instruments for measuring and analyzing carbon dioxide concentration in incubators. Fyrite absorbing fluid is selective in the chemical absorption of carbon dioxide (Figs 16A and B).

pH Meter: This is used to measure pH.

Ultra pure water system: A water filtration system should be installed to provide clean water for washing the laboratory or we can use double distilled water (Figs 17A to C).

The database systems are developed to manage patient and laboratory information and management of internal quality control and external quality assurance systems for andrology and embryology. There are many software programs for this purpose, which are commercially available today. A user-friendly one should be chosen that could be used widely. Besides containing essential details of the patient's records, it must contain history of the cause of infertility as diagnosed earlier, results of new diagnosis if relevant, the treatment option best suited for the particular patient, the treatment carried out and the outcome of treatment and follow-up, if any. Any other noteworthy point such as possible adverse reaction to drugs must be recorded.

Quality of consumables used in the laboratory: All disposable plastic ware must be procured from reliable sources after ensuring that they are not toxic to the embryo. They are sterilized by gradiation and individually packed. Box 1 shows a list of consumables needed (Figs 18A and B).

When starting an IVF laboratory, it should be ensured that culture media and mineral oil used for processing gametes or growing embryos in vitro should be procured from reliable manufacturers. The embryologist should know the composition of the media that are being used. Each batch of culture medium needs to be tested for sterility, endotoxins, osmolality and pH to ensure that it is of tissue culture grade, preferably mouse embryo tested and with purity appropriate for the purpose. It is important to check that producers use validated quality control testing, if not this has to be done by the laboratory. Packaging and appropriate delivery conditions should be ensured. Expiry date should be recorded. Most media are supplemented with serum; they should, therefore, be tested for antibodies to HIV 1 and 2, hepatitis B surface antigen and hepatitis C RNA. Each lot of culture media and mineral oil used should be recorded in each patient's worksheet. The media used are flushing medium, sperm preparation medium, universal IVF medium, mineral oil and embryo freezing and thawing package (Fig. 19).

Protocols in the laboratory must be set up before starting. All consent forms should be in order for each procedure. The detailed manuals for all procedures used should be available in the laboratory. Written, signed and dated protocols and standard operating procedure should exist for every process performed in the laboratory. Proper training of all the laboratory staff accordingly to the procedures followed and the laboratory is mandatory. The laboratory set up must include provision for unique patient identification, and corresponding gametes, zygotes and embryos identification, while retaining patient confidentiality. All samples from the patients, like blood, follicular fluid and sperm samples, must bear identification of the treated couple. A system of checks and where needed, double checks by a second person must be established. Incubators should be organized in order to facilitate identification of embryos, zygotes, oocytes and sperm. Patient's identity must be verified and recorded before ovum pick up, at semen recovery, at insemination or ICSI, at cryopreservation and at embryo transfer procedures. Documentation of all critical steps in each patient's file is essential. The identity of the laboratory person handling the samples at each point of the process, from receipt through final disposition, date and time, should be clearly indicated.²⁰ Strict observation of staff cleanliness is essential. Food, drinks and cigarettes are strictly forbidden. The use of make-up and perfumes should not be allowed. Washing hands and change of clothes while entering the laboratory is important.

Quality control: Quality management system must be in place. A written record of adverse events, emergency 18situations and incorrect identification of specimens is maintained. Laminar flow hoods, laboratory tables, incubators and other areas where sterility is required must be periodically checked for microbial contamination using standard techniques, and a record of such checks must be kept. A logbook should be maintained, which records the temperature, carbon dioxide content and humidity of the incubators and the manometer readings of the laminar air flow. All instruments must be calibrated periodically (weekly, monthly or yearly) and a record of such calibration maintained.²⁰

Risk management: Before starting the laboratory each unit should establish procedures and policies for the safety of personnel and for preventing cross contamination. All personnel must be vaccinated against hepatitis B or other viral disease. Screening patients and gamete donors for human immunodeficiency virus (HIV), hepatitis B/C should be the norm for the laboratories. The laboratory staff must be educated to treat each sample as potentially infectious and must be made aware about the risks of handling infected biological material. Care must be taken for the safe disposal of biological waste and other materials (syringes, glass slides, etc.).