cairo consensus:a groundbreaking guide for improving ART lab design, air quality, and IVF success rates

Discover the key highlights of the Cairo Consensus—a groundbreaking guide for improving ART lab design, air quality, and IVF success rates. Perfect for embryologists and IVF professionals!

1. Cairo consensus Dr REHA RAKHOLIA Mch REPRODUCTIVE MEDICINE AND SURGERY lecture in youtube link---> https://www.youtube.com/watch?v=EouqiH2zmaE

2. Introduction Increasing regulation and licensing of IVF laboratories, especially for non-hospital surgical facilities. New regulations impose hospital-level infection control standards, not considering the unique requirements of assisted reproduction technology (ART) laboratories. Consensus Workshop-Held at Upper Egypt Assisted Reproduction Symposium (UEARS) 2017 in Cairo, Egypt. Goal: Establish technical and operational requirements for air quality in IVF Centers . Set aspirational benchmarks for existing ART laboratories. Provide guidelines for the construction of new ART laboratories.

3. Challenges with Current Regulations Air-quality requirements developed as if IVF suites are surgical facilities. This fails to consider the need for minimal physicochemical stress on gametes and embryos. ART laboratories require more specialized environmental controls than traditional hospital environments.

4. Workshop Focus Areas & Safe Practices Design and construction of the HVAC ( heating, ventilation and air conditioning system) system for IVF labs. Control of particulates, micro-organisms , and VOCs in critical lab areas. Selection of construction materials to minimize VOCs and contaminants. Safe cleaning practices to protect gametes and embryos from toxins. Operational practices to optimize air quality, balancing temperature control and airflow. Infection-control measures to reduce VOC exposure (e.g., cold sterilizers, surface cleaners, hand sanitizers).

5. How the laboratory environment affects gamete and embryo biology

6. Laboratory Environment and IVF Process IVF success is governed by the biology of gametes and embryos. Lab design, HVAC systems, and equipment materials must align with biological needs. Primary goal: Protect gametes and embryos from adverse external factors. Adaptation to stress affects embryonic gene expression, imprinting, and epigenetic inheritance.

7. Factors Affecting IVF Outcomes Patient Biology: Affects the quality of gametes and embryos. Clinical Factors: Stimulation, oocyte retrieval, embryo transfer, and luteal phase support impact outcomes. Laboratory Environment: Equipment, contact materials, methodology and staff are key influencing factors.

8. Environmental Factors Micro Scale: Temperature control, oxygen, and CO2 levels (pH balance) affect embryo development. Macro Scale: Laboratory design and air quality significantly influence outcomes. Regular equipment maintenance, verified calibration, and preventing malfunctions are essential.

9. Contact Materials and Methodology Materials such as culture medium, plasticware, gases, handling devices, intracytoplasmic sperm injection microtools, and oocyte retrieval needles and embryo transfer catheters influence embryo viability. Oocyte Retrieval and Handling: Stable temperature during follicular aspiration, transport, and handling is crucial. Methodology: Proper technical approaches and adherence to standard procedures ensure optimal outcomes.

10. Impact of Temp and Gas Stable Temperature Maintenance: Maintaining stable temperature during follicular aspiration, transport, egg search, and cumulus–oocyte complex handling is crucial for oocyte functionality (Mortimer & Mortimer, 2015). Gas Equilibrium for Oocytes and Embryos: Stable pCO2 is required for bicarbonate-buffered medium pH equilibration, while reduced pO2 protects against oxidative stress . Zwitterion-buffered media with sufficient bicarbonate should be used to support embryo metabolism when handling under air (Blake et al., 1999).

11. Impact of Microenvironment Changes Even small changes in the microenvironment affect oocyte metabolism. For ex, a 5-minute exposure of fertilized mouse oocytes to a medium without amino acids reduced the number of blastocysts formed and cell counts (Gardner & Lane, 1996).

12. Protection of Gametes and Embryos from Toxic Substances Toxic Substances : VOCs (Volatile Organic Compounds) and airborne chemicals can harm gametes and embryos. Lab Design & Materials : Ensure safe design, choice of non-toxic building materials (paints, adhesives, sealants). Workstations & Incubators : Select systems that protect from harmful exposures. Gas Supply : Use purified gas and well-designed gas supply systems. Potential Air Quality Issues : Construction materials: paints, adhesives, sealants. HVAC system air quality. Cleaning products and air fresheners. VOCs from lab equipment. Human contaminants: cosmetics, hair, skin cells. Clothing fibres, laundry products, sanitizers for infection control.

13. Evidence in clinical medicine: Harper Model (2012): Six steps for introducing new technology in IVF labs: Hypothesis-driven research Testing in animal models Testing with donated embryos Small-scale pre-clinical investigations Larger clinical trials Assessment of clinical and cost-effectiveness

14. The HVAC system and cleanroom standards Historical features of ART laboratory design Design philosophy for a new ART laboratory suite Physical isolation criteria Retrofitting existing laboratory suites

15. Historical Features of ART Laboratory Design (Pre-2000) Labs designed like surgical suites or medical offices without consideration for cell culture needs.Common design issues:Vinyl flooring, MDF casework, and shared HVAC systems. No air handling systems or dedicated gas supply room. Cryopreservation areas without proper floor protection. No entry vestibules (airlocks), and IVF labs separated by hallways. Introduction of air showers and ultra-low particulate filtration towards the late 1990s.

16. Improvements in ART Laboratory Design Post-2000 Introduction of HEPA filtration, laminar flow hoods, and positive pressure systems. Segmentation of labs: separate culture suite, diagnostic andrology, and gas storage. Improved air handling units (AHU) and dedicated air ducts to prevent contamination.

17. AIA Guidelines for ART Laboratories American Institute of Architects–Department of Health and Human ServicesGuidelines (1996-1997, updated in 2001) : No specific requirements for ART labs. Most stringent standards based on surgical rooms: Positive pressure air movement . Minimum 15 air changes per hour (ACH) , 3 fresh air changes (FACH). No recirculating air units (e.g., no window AC units). Temperature: 20–23°C (68-73°F) , humidity: 30-60% . Lower humidity risks eye irritation, static electricity; higher humidity risks mold growth.

18. Current Guidelines and Changes (Europe/Asia) Focus on particle removal but recognizing that this alone is insufficient. Segmented lab design: Separate diagnostic andrology, endocrinology, culture suite . Separate gas room and entry vestibules to minimize contamination. Improved air quality control: Use of HEPA filtration , dedicated air handling unit (AHU), and chemical filtration. Positive pressure systems to prevent contamination from outside air.

19. Common Design and Operating Issues in IVF Labs Incomplete Isolation: Lack of complete separation from surrounding areas. Inadequate Positive Pressure: Insufficient pressurization allows ‘dirty’ air to flow into the lab. Chemical Filtration Issues: Incorrect filter media (e.g., activated charcoal alone). Incomplete oxidation of some VOCs by photochemical systems. Failure to monitor and maintain filtration systems. Inappropriate Construction Materials: Use of MDF, linoleum flooring, oil-coated ductwork, and formaldehyde-urea insulation. Low Fresh Air Ventilation Rates. HVAC System Bypass: Air can bypass filters due to poor pressure monitoring or lack of proper maintenance. Compromised Air Intake: Air intake placed in contaminated areas, such as shipping docks. Difficult Maintenance: Poor design (e.g., AHU or chemical filter placed in ceiling space). Inadequate Humidity Control.

20. Design philosophy for a new ART laboratory suite Define Functions of ART Suite: Retrieval room Transfer room IVF culture and biopsy lab Cryopreservation space attached to the suite Use of Double-Door gasketed Pass-Through Windows: Maintains positive pressure Reduces air leakage between IVF lab and procedure rooms Hard Lid Ceiling: Required in surgical and clinical rooms to control air leakage

21. Separation of 'Dirty' Rooms Dirty Rooms to be Separated from ART Suite: Gas cylinder rooms Storage rooms Fixation and staining rooms Diagnostic andrology and endocrine assay labs Staff offices, changing rooms, janitorial closets Cryobank (due to continuous ventilation needs)

22. ART Suite Clean Environment - Key Measures Control Outside Infiltration Use vestibules or staged interlocking doors. Lighting mounted on the ceiling (not in). Gasketed access panels, sealed with silicone. Re-seal access panels after each opening. Pressure Monitoring Routine monitoring of pressure across IVF lab and AHU. Maintain a positive pressure environment. Ductwork and AHU Components Ensure no rust inhibitors (source of VOCs).

23. Gasket Silicon sealent

24. Gas and Ventilation Management Gas Rooms & Lines Separate rooms for compressed gases/liquid nitrogen. Make sure gas lines are sealed with extra protection where they pass through the walls, to prevent any leaks. Fire Protection Regulations: Apply to gas and cryobank rooms (contain volatile liquids). Firebreak Dampers: Avoid installation in ventilation ducts. Risk of anoxia (oxygen deficiency) or CO2 intoxication if ventilation dampers close without alternative air supply.

25. When smoke or fire is detected, the damper closes, blocking air from flowing through the duct to limit the spread of fire and smoke to other areas.

26. Physical Isolation Criteria Physical isolation : Secure sealed barriers (walls, doors, ceiling) and high positive pressure to prevent contaminants. Sulphur hexafluoride infiltration studies measure isolation effectiveness.(SF6 infiltration studies check if a lab is properly sealed by releasing gas outside and seeing if it leaks in, helping to keep the lab free from contamination.)

27. Retrofitting Existing Laboratory Suites Common strategies to improve existing labs: Install boost fan for higher positive pressure. Use double-door pass-through between IVF lab and procedure rooms.( if procedure room is not as clean as ivf lab ) Reduce new materials directly brought into the lab. Use supplementary air cleaning systems to lower particulates.

28. Controlling VOCs: the fabric of the laboratory Measuring VOCs and aldehydes Sources of aldehydes in ART laboratory settings

29. What are VOCs? Volatile Organic Compounds : Organic chemicals that evaporate at room temperature. Sources: Outgassing from lab materials (furniture, consumables), gases used in incubators, and contaminants in culture media or mineral oil. How VOCs Spread: VOCs can move between different mediums (air, water, oil). This movement depends on factors like concentration, temperature, and pressure. 'Sink' Effect : A medium (e.g., water) acts as a reservoir, absorbing VOCs from another medium (e.g., air).

30. Non-Toxic VOCs Examples: Silicones : Found in incubator gaskets and tubing (stable, non-reactive). Paraffinic Oils : High molecular weight oils (non-toxic, stable). d-limonene and α- pinene (found in colognes/ cleaners ) – Oil-soluble but not reactive or harmful under culture conditions. Biologically harmful VOCs : Ethanol (metabolized into acetaldehyde) and isopropyl alcohol (metabolized into formaldehyde). Key Principle : Even non-toxic VOCs should be minimized to reduce risk. For harmful effects, VOCs need to, have high vapor pressure , be soluble in culture media or mineral oil , and be reactive

31. Factors That Modulate Contaminant Exposure: Pollution Control Technology : The ability to remove or reduce contaminants using filters and air systems. Contamination Routes : How contaminants enter the system (air, culture media, gas supply, or lab equipment). Absorption or Release : Materials in the environment can absorb or release contaminants, affecting exposure. Chemical Reactions : Contaminants can react with substances like oils, media, or biological materials, possibly making them more or less harmful.

32. Penetration of Contaminants : Contaminants can reach and affect sensitive materials like embryos, cells, or lab cultures. Biological Impact : Gametes, embryos, or zygotes can be affected directly by contaminants. Detoxification : Biological systems like gametes, zygotes, embryos, and blastocysts have the capacity to metabolize contaminants. They can detoxify, excrete, or sometimes transform contaminants into less harmful or more toxic forms using enzymes such as cytochrome P450. Solubility and Reactivity : Changes in solubility (in air, water, oil) can shift how contaminants move through the system. Advanced Filtration : HVAC systems and gas filters help lower contamination levels in laboratories.

33. Measuring VOCs and Aldehydes in ART Labs Olfactory Method (Smell): Unreliable for detecting VOCs due to individual differences in sensitivity. Odor threshold may be higher than toxicity levels. Photoionization Detectors (PID): Measures VOC levels in ppm (mg/m³) to ppb (µg/m³) range. Cannot identify specific VOCs; poor for aldehydes. Useful for checking filter performance (e.g., activated carbon). Gas Chromatography (GC): Identifies and quantifies 65-75 VOCs (EPA TO15 method). Detects VOCs at low levels ( µg/m³ or ppb ). Can semi-quantify uncalibrated materials.

34. Sources of Aldehydes in ART Laboratory Settings Impact on Embryo Development : Elevated levels of formaldehyde, acetaldehyde, higher molecular weight aldehydes Reduction in aldehyde levels linked to poor, delayed, or no embryo development. improved blastocyst development

35. Common Aldehydes in ART Laboratories Common Contaminants : Formaldehyde Acetaldehyde Propionaldehyde Butyraldehyde Benzaldehyde n- Hexaldehyde Acrolein Risks :Known carcinogens and mutagens (Hazardous Substance Data Bank). Another common compound, acetonitrile, has also been suggested as a possible source for the slow release of cyanide.

36. Sources of formaldehydes in ART Labs Environmental Sources : Photochemical smog (high solar flux areas like California, Texas, Guatemala). Incomplete combustion (gas water heaters, motor vehicles). Internal Sources : MDF cabinetry ( Medium-density fiberboard ) : Formaldehyde off-gassing, especially at higher temperatures. Compressed CO2 : Aldehyde residues in compressed gas tanks (e.g., acetaldehyde,isovaleraldehyde , benzaldehyde,formaldehyde ). If liquid CO2 is present in the cylinder, they will remain dissolved and not enter the gas phase, but when the pressure in the gas tank becomes low, the aldehydes will enter the gas phase and pass into the incubator. Cold sterilization ( eg - cidex solutions ) : Use of glutaraldehyde. Pathology biopsy fixation (10% formalin). New plasticware packaging : Styrene release.

37. Avoiding VOCs in culture Avoid introducing VOC into the laboratory Decrease ambient VOCs in the laboratory Decrease VOCs in incubators

38. Intro 1. Optimizing Culture Environment: .

39. 2. Risks from External Exposure:

40. 3. Prevention of Contamination:

41. Ultraviolet Light Photooxidation for Air Purification

42. VOC Exposure and Impact on Embryo Development: affect embryo development from the early stages of culture and potentially have long-term effects after implantation. Culture conditions - influences the appearance of embryos during culture and their performance after culture

43. Avoid introducing VOC into the laboratory

44. Painting the Laboratory Standard paints can release VOCs . Use low-VOC or VOC-free paints to reduce toxicity. Avoid adding pigment at the point of sale to prevent increasing VOC content. Inform building personnel about the effects of VOCs on embryos. Carry out painting when no embryo culture is in progress. Speed up VOC release after renovation by increasing room temperature and turning on lights preferably days or weeks .

45. Selection of Laboratory Furniture MDF cabinets release formaldehyde . Older wooden furniture might have off-gassed and be less harmful. Stainless steel is less likely to emit VOCs but should be cleaned with isopropyl alcohol to remove any superficial VOCs before introduction into the laboratory. Use silicone-based lubricants for hinges and drawer slides to avoid VOCs from other greases.

46. Incubator Commissioning Plastic gaskets around incubator doors may release embryotoxic substances . New Incubators should be off-gassed whenever possible, e.g. running new units in a ventilated space (not the embryology laboratory) at high temperature for some time(1–2 months ). After major repairs , ensure parts do not introduce new VOCs. Off-gassing is the process of releasing harmful gases before use. .

47. Off-Gassing Plasticware Polystyrene plasticware releases styrene . Off-gas plasticware outside the lab by opening sleeves in a separate room. Maintain sterility during off-gassing to avoid contamination. Individually packaged plasticware (like multi-well dishes and pipettes) makes it harder for VOCs to be released. For individually packaged plasticware a solution is to open all the dishes needed for the week and place them in a laminar flow hood, allowing VOCs to escape. Plasticware packaged in filtered sleeves has fewer VOCs because the sleeves let gases escape before you open the package

48. Managing HVAC Air Intake HVAC system should reduce VOCs. Outdoor air may contain high VOCs due to: Roofing repairs or tar replacement (fresh air inlet on the roof) Exposure to smoke from large fires in the region Parking lot repaving or painting near the building Nearby construction using heavy diesel equipment Open holes in the ground near the lab Solution: Switch to 100% air recirculation during high VOC events and monitortemperature / humidity.

49. Potential Consequences of 100% air Recirculation:

50. Smoking and Laboratory Safety Smoking near the lab or HVAC air intake can introduce embryo toxins. Third-hand smoke: Personnel who smoke can bring toxins into the lab via clothes, skin, and breath.

51. Cleaning Practices in the Lab Ethanol is commonly used to provide a clean and relatively microbe-free work surface,but is a VOC and can be toxic to embryos. Alternatives: Use dilute sodium hypochlorite ( chlorox )or hydrogen peroxide. Some labs prefer water cleaning to avoid embryotoxicity.

52. Toxicity from Other Sources Potential toxins from: - Detergents for laundering scrubs - Solvents in labeling pens - Personal care products (e.g., shoe polish, hairspray, deodorants) Weigh the benefits of using these products against the risks.

53. Decrease ambient VOCs in the laboratory Despite best efforts, VOCs (volatile organic compounds) will be present in the laboratory. Two systems to help reduce VOC levels: HVAC system In-room air purifiers Carbon filtration and ultraviolet photocatalytic oxidation reduce VOCs; HEPA filters do not( they reduces particulates).

54. HVAC System for VOC Reduction in Labs 1. Regulates temperature, humidity , and provides clean air. 2. Key Components: HEPA Filters : Remove particulates (bacteria, mold spores). VOC Removal Technologies : Activated Carbon with Potassium Permanganate. Ultraviolet Photocatalytic Oxidation . 3. Filter size and type must match the laboratory's air volume to ensure proper air turnover. Humidity control is crucial: too high - mold growth, too low humidity- desiccation (drying out) of the culture medium,. 4. Maintenance: Regular filter changes to ensure proper air turnover. Monitor Humidity to avoid harmful extremes.

55. In-Room Purifiers: In IVF labs, HVAC systems use activated carbon filters to clean the air by removing VOCs .However , carbon filters alone can't remove certain other chemicals. That's why potassium permanganate is used alongside carbon filters, providing an additional layer of protection to remove these harmful chemicals and keep cultures and embryos safe. Portable devices can reduce VOCs but are less effective than HVAC systems. Require regular filter changes for effectiveness.

56. Decrease VOCs in incubators 1) Purchased Gases: Ensure high-purity medical grade or reagent grade gases for incubators. Discuss purity standards with suppliers to avoid embryotoxicity. 2)In-Line Filters: Use carbon and potassium permanganate(strong oxidizing agent) filters. Activated carbon -Adsorbs VOCs onto its porous surface, trapping them physically. Potassium permanganate( redox agents)- Oxidizes VOCs into non-toxic byproducts (e.g., CO₂, H₂O). Replace filters regularly to maintain air quality.

57. Redox agents oxidize VOCs and other harmful substances present in the air, breaking them down into less harmful compounds

58. 3) Incubator Type: Big-box incubators( Traditional incubators) use room air, increasing VOC exposure. Modern incubators (Benchtop incubators ) use cylinder-based gases, reducing room air VOCs. As the incubator flushes the chamber(s) with the mixed gas, any ambient room air is flushed out of the chamber, thereby leaving the tanks and in-line filters as the only source of contaminants inside the incubation chamber.

59. 4)Intra-Incubator Recirculating Filters These filters are placed inside large incubators (old-style ‘big-box’ incubators) to reduce harmful VOCs inside the incubator. Gases in the incubator are recirculated through a carbon filtration system to remove VOCs. Time-Lapse and Benchtop Incubators : These incubators continuously mix gases and recirculate them through an additional carbon filter. This doubles the filtration compared to systems that only filter gases in the gas line. Important Condition : Effective only when the atmosphere is not humidified . Water on the surface of carbon filters reduces their ability to remove VOCs.

60. Traditional big box Incubator – Intraincubator filter

61. Benchtop Incubator- filter

62. 5) Decrease VOCs in cultures VOCs can infiltrate from gases inside incubators or brief exposure to room air. Once VOCs enter the culture, removing them is difficult, but their concentration can be reduced. Types of VOCs : Hydrophobic VOCs : e.g., benzene, styrene—prefer oil-like phases and can easily dissolve into the embryos' membranes, which are made of lipid layers. This can disrupt the membrane's function and harm the embryo. Hydrophilic VOCs : e.g., ethanol, formaldehyde—dissolve easily in aqueous phases, including embryo cytoplasm.

63. Types of VOCs : Hydrophobic VOCs : e.g., benzene, styrene—prefer oil-like phases and can easily dissolve into the embryos' membranes, which are made of lipid layers. This can disrupt the membrane's function and harm the embryo. Hydrophilic VOCs : e.g., ethanol,acrolein formaldehyde,glutaraldehyde —dissolve easily in aqueous phases.

64. Managing VOCs with Oil Overlay Oil overlay is commonly used in IVF labs to prevent the loss of carbon dioxide. ( negligible benefit as it slows down medium re-gassing.) Stop drying (desiccation) of culture media, keep the temperature stable when cultures are taken out of the incubator. Detoxification of Culture -Oil overlay helps reduce hydrophobic VOC levels in the culture medium as VOCs tend to partition into the oil at higher concentrations, diluting them in the oil phase, thus lowering VOC concentration in the aqueous phase. Acts as a partial barrier (not an Absolute Barrier): VOCs can still enter the culture medium if exposed for long periods

65. Hydrophobic VOCs: These chemicals prefer oil over water. Oil overlay helps remove these VOCs from the water-based culture medium, reducing their concentration. Dilution Effect: Since the oil volume is much greater than the medium, VOCs get diluted in the oil.

66. Hydrophilic VOCs Oil also slows down the movement of hydrophilic VOCs preventing damage of the embryos during short exposure periods. These chemicals dissolve better in water than oil, so even with oil overlay, they can eventually reach the culture medium if exposure is long enough.

67. Surface Area,volume and Oil Exposure Large Surface Area : When the oil layer in culture dishes is spread out, it can absorb more harmful chemicals (VOCs) from the air. This happens because more oil is exposed to the air. Small Surface Area : If the oil layer is deeper and covers a smaller area, it absorbs fewer harmful chemicals because less oil is exposed to the air. Dilution Effect : A larger volume of oil can dilute the VOCs more effectively, reducing their concentration even further in the culture medium. This helps protect the embryos.

68. Oil Overlay in Practice Use of Oil Sinks: To further reduce VOC exposure, blank dishes filled with oil can be used as ‘oil sinks,’ absorbing VOCs from the air before they reach the culture. Impact on CO2 and pH: Oil overlay can slow down the exchange of carbon dioxide , which can affect the pH of the culture medium. This is why pre-incubation is often needed to ensure the culture medium reaches the correct pH before being used for embryo cultures.

69. Consensus points

70. INTRO : Cleanroom Design for ART Labs ART labs should have air quality comparable to ISO Class 6 or GMP Grade B/C , too many air changes per hour (ACH) can cause overcooling of embryos , which is harmful.

71. Middle Ground: The group decided that ISO Class 7/GMP Grade B (in operation) and Grade C (at rest) would be the target. This can be achieved with HEPA filters and 10–15 air changes per hour . Procedure Rooms: IVF procedure rooms (used for tasks like oocyte retrieval or embryo transfer or percutsneous sperm retrieval ) should be different from surgical rooms , with a separate HVAC system from art suite if surgery is involved. Mobile Air Filtration :Can be used to achieve air quality standards at rest but does not provide positive pressure Grade At Rest (0.5 µm) At Rest (5 µm) In Operation (0.5 µm) In Operation (5 µm) B 3,520 29 352,000 2,900 C 352,000 2,900 3,520,000 29,000

72. 1. Assessing site suitability Location: Choose a location away from areas with heavy pollution like car garages, dry cleaners, factories, and gas stations. These places can release harmful substances into the air, which can affect the delicate processes in an IVF lab. VOC Analysis : Test for volatile organic compounds (VOCs) in and around the building. VOCs can evaporate into the air and harm embryos, so maintaining safe levels is crucial

73. Local Air Quality : Review local data on particulate matter (PM5 and PM10) . These are tiny particles suspended in the air, with diameters up to 5 micrometers (PM5) and 10 micrometers (PM10) . These particles pose risks to embryo development and lab integrity. Monitor nearby pollution sources (e.g., parking garages, dry cleaners, foundries, petroleum processing facilities). Implement measures to reduce particle ingress to ensure a clean, safe environment for IVF procedures. .

74. Basic design criteria (new construction)

75. Air quality The ART lab should have HEPA-filtered air quality equal to that of an operating room. No Criteria Details 2 Particulates Less than 352,000 particles larger than 0.5 μ m to 10 μ m per m³ (equivalent to <10,000 such particles per cubic foot). 3 Air Quality (HEPA Filtered) The ART lab should have HEPA-filtered air quality equal to that of an operating room. 4 Micro-organisms Less than 10 colony-forming units ( cfu ) per m³ and less than two spores per m³ ‘at rest’. 5 VOCs Total VOCs: Must be <500 μ g/m³ (~ 400–800 ppb depending on molecular species ) to prevent embryo/gamete damage. Aldehydes: Must be <5 μ g/m³ as they are highly toxic.

76. Hepa filter Voc’s Particles Micro organism

77. 6 Air Changes Fifteen total air changes per hour, including three fresh air changes per hour (20% outside air). Consider VOC filtration type and manufacturer’s instructions regarding air changes. 7 Overpressure Target overpressure of +38 to +50 Pa in the IVF lab (minimum recommended +30 Pa), achieved through a cascade of overpressure across rooms to manage pressure differences. 8 Temperature Maintain a stable working temperature between 20–24°C , controlled by HVAC, considering heat output from lab equipment and staff to ensure staff comfort and equipment stability. 9 Humidity Relative humidity between 40% and 45% to prevent mold growth, ensure comfort, and avoid high evaporation rates that could affect the osmolarity of the culture medium and harm embryos.

78. humidity pressure Air changes Temperature

79. 10. Sealing Ensure the lab is sealed with the specified materials. Daily inspections are recommended. 11. Activated Carbon/Potassium Permanganate Filter Add a carbon or permanganate filter to remove VOCs, placed before the HEPA filter, with a 0.2–0.35 sec residence time. 12. Heating and Cooling of Incoming Fresh Air Make sure the system heats or cools fresh air as needed based on local climate. 13. Isolating External Air The HVAC system should be able to fully block outside air during emergencies (e.g., fires, high pollution, nearby construction). In this "submarine" mode, over-pressure may be lost , but it protects the lab from harmful air.

80. Heating and Cooling of Incoming Fresh Air Isolating External Air Sealing Activated Carbon/Potassium Permanganate Filter

81. 14. Air Supply Vents and Return Ducts Place air supply vents in the ceiling and return ducts near the floor. Avoid drafts that could disrupt equipment. 15. HEPA Filters Should be located centrally to avoid the need for access to multiple locations within the ART suite when changing them. 16. Air Quality To achieve optimum air quality, the system should be recirculating with only 20% fresh air to create necessary over-pressurization. 17. Pressure Sensors Pressure sensors and differential pressure displays should ideally be installed on each side of each doorway into, out of, and between areas within the ART suite.

82. Types of filters. 1. HEPA (High efficiency particulate air) filters 2. ULPA (ultra-low penetration air) filters 3. VOC (volatile organic compound) Filters

83. Types of filters. HEPA (High efficiency particulate air) filters ULPA (ultra-low penetration air) filters 3. VOC (volatile organic compound) Filters

84. Feature HEPA Filter ULPA Filter Efficiency 99.97% (≥ 0.3 microns) 99.999% (≥ 0.12 microns) Particle Size ≥ 0.3 microns ≥ 0.12 microns Applications General clean environments Ultra-clean environments Airflow Resistance Lower Higher Cost Moderate High Maintenance Less frequent More frequent

85. Sealing the box’ Effective over-pressure must be achieved by building the cleanroom suite to minimize air loss, ensuring air flows out of the embryology laboratory. 18 Slab-to-Slab Walls Exterior walls of the cleanroom must go from the concrete floor to the underside of the floor above (slab-to-slab) with all perforations completely sealed . 19 Ceiling Should be composed of a solid, contiguous material (e.g., plasterboard, gypsum panels, Gyprock, Sheetrock®) with minimal access panels. Essential panels must have air-tight, silicone gaskets. 20 Light Fittings Must be air-tight, designed for cleanrooms to prevent air leakage into the plenum void above. Surface-mounted fittings are acceptable if cable access is sealed and there are no horizontal rims or flanges. 21 Electrical, Gas, and Data Conduits All conduits should be sealed where they enter or leave the cleanroom to prevent air loss. Use steel ‘Dado’ trunking within the suite for distribution of power, data, and gas lines. 22 Doors Doors must be tight-fitting with bottom sweeps and perimeter seals (top and edges). Any view panels must be mounted using gaskets to ensure they are air-tight. 23 Pass-Throughs Must be air-tight to maintain room air pressure differentials.

86. Lightning Ceiling wall Vinyl flooring

87. Construction materials No. Component Details 24 Walls True cleanroom modular panels with powder-coated metal, gasketed interfaces, or plasterboard coated with zero VOC paint. 25 Floors Sheet vinyl with impervious sealed joints; stainless-steel tread plate in areas with liquid nitrogen. 26 Countertops Non-porous materials like epoxy, Corian®, and Trespa ® to prevent VOC release ensure non-porous, easy-to-clean surfaces. 27 Ceilings Gasketed and sealed inspection or access panels, minimized panels to maintain cleanroom standards. 28 Windows Glass, gasketed, and small, mounted in doors; spectral filters for outdoor windows to exclude UV light. Large observation windows require proper seals. 29 Cabinets Powder-coated metal or stainless steel for durability , non-toxicity, and contamination resistance. 30 Wood Products Avoid materials like MDF, Formica®, linoleum, or oil-based paints due to embryotoxicity.

88. stainless-steel tread plate in areas with liquid nitrogen

89. Plumbing No. Component Details 31 Handwashing Facilities Should be located in vestibules rather than in laboratories. Waste pipes require traps. 32 Noxious and Corrosive Chemicals These should not be used in IVF laboratories, eliminating the need for drench showers. If required by local codes, ensure traps do not dry out. 33 Fire Suppression System Fire suppression systems (e.g., sprinklers) should be protected from accidental triggering from outside the laboratory. 34 Open Flame If open flames are used, conduct a risk assessment for fire likelihood and exposure to combustion products. 35 Plumbing Avoid unnecessary plumbing in ceiling plenum spaces as it poses risks of flooding and contamination, especially during renovations.

90. Laboratory commissioning and ongoing VOC management

91. 36 Burn-in Newly constructed or renovated laboratories should allow 2–3 weeks minimum for off-gassing of construction materials, verified by specific VOC testing . Bioassays like human sperm survival tests are not sufficient. 37 Deep Clean Laboratories must undergo intensive cleaning of all surfaces, including hard-to-reach corners, cupboards, drawers, and equipment. Cleaning products should remove all contaminants, and no cleaning agent residue should remain . 38 Servicing A cleanroom HVAC system must be serviced annually or more frequently if performance fails to meet expectations. Pleated particulate filters should be changed every 90 days , and HEPA filters typically last 2–4 years . Pressure differentials across filters should be monitored using gauges like Magnehelic ®. Chemical Filter Replacement: Changing chemical filters at set intervals is not optimal . A facility's filter consumption history should guide replacement schedules. Excessive solvent use, fires, or construction activities can rapidly exhaust filters.

92. Pleated filter HEPA filter Activated carbon filter Magnehelic gauge

93. Types of Filters Pleated Filters : Capture larger particles like dust and debris. HEPA Filters (99.97%) : High-efficiency filters that remove ultra-fine particles, including allergens, bacteria, and some viruses. Chemical Filters These remove chemical contaminants and odors by adsorbing gases or vapors .

94. 39 Infection Control Infection control measures used in hospitals might be embryotoxic. Products like hand sanitizers must be evaluated before being introduced into the ART suite. 40 Aldehyde-Based Cold Sterilizers Aldehyde-based sterilizers must not be used within the IVF suite due to embryotoxic vapors . Glutaraldehyde stations must be located outside areas where vapors could contaminate the suite. Cleaning Agents Cleaning agents containing chlorine dioxide (bleach) are not appropriate. Cleaning with 6% hydrogen peroxide (H₂O₂) is recommended as it oxidatively destroys microorganisms and is easily removed. Alcohols (70% ethanol or isopropyl alcohol) are less effective as they only denature proteins. Alcohol-Free Cleaning Alternatives Alcohol-free alternatives, such as quaternary ammonium compound-based cleaners , are considered safe and can replace traditional alcohol or bleach-based cleaners. Care must be taken to ensure embryos are not exposed to residues of these cleaning agents.

95. Quaternary ammonium compound: Safe disinfection for CO2 incubators, laminar flow hoods, glass, plastic, metal, Hi-Macs surfaces, equipments , ultrasound probes and hospital furnitures ORDER INFORMATION OODIH-5000 Oosafe ® Disinfectant for CO, Incubators and Laminar Flow Hoods, 5L refill OODIH-1000 Oosafe ® Disinfectant for CO, Incubators and Laminar Flow Hoods, 1L with spray OODIH-0050 Oosafe ® Disinfectant for CO, Incubators and Laminar Flow Hoods, 50ml with spray OODW-70 Oosafe * Disinfection Wipes, 70 wipes/bucket

96. Quaternary ammonium compound : Safe disinfection and cleaning for laboratory surfaces and floor. It is also compatible with glass, metal, rubber, plastic and wood. ORDER INFORMATION OODSF-10000 Oosafe ® Surface Disinfectant, 10L refill OODSF-02000 Oosafe ® Surface Disinfectant, 2L with dose cup OODSF-00050 Oosafe ® Surface Disinfectant, 50ml

97. Aspect Oosafe ® disinfection for CO2 incubators, laminar flow hoods, glass, plastic, metal, Hi-Macs surfaces, equipments , ultrasound probes and hospital furnitures Oosafe ® Surface Disinfectant Microbiological Efficacy Effective against Hepatitis B, HIV, Rota virus within 1 minute , and against bacteria, fungi (Candida) , and Influenza A virus (H5N1/H1N1) within 15 minutes . Effective as a bactericide, fungicide (Candida), algicide , and selectively virucidal . Application Instructions Apply evenly on surfaces using a spray or wipe . After application, wait 15 minutes and clean with a humid sterile sponge or cloth to remove residues. Dilute the solution with water (1:100) before application. After application, wait 15 minutes , and rinse the surface if used on the floor (to prevent slipperiness).

98. 41 Plasticware Plasticware off-gasses VOCs, especially polystyrene, which releases embryotoxic styrene. All plastic cultureware should be off-gassed outside the laboratory (while maintaining sterility) . Manufacturers are encouraged to use breathable packaging . 42 Packaging Materials Packaging exposed to the outdoor environment, like cardboard, must not enter the ART suite as it can introduce fibres, dirt, fungal spores, and particulates. Minimize the use of interior cardboard and paper packaging in the ART suite. 43 Cleaning and Cosmetic Products Avoid using cleaning products, air fresheners, cosmetics, perfumes, aftershaves, nail polish, handwashing products, and hand sanitizers that release VOCs. 44 Perfume-Free Environment Clinical areas in the ART suite should operate as perfume-free zones. Patients must avoid using skin care products, cosmetics, and grooming products when entering these areas.

99. 45 Third-Hand Smoke Prevent contaminants from ‘third-hand’ smoke (residue on hair and clothing from smoking or smoke exposure) from entering the IVF laboratory or ART suite. 46 Operators as Sources of VOCs Operators themselves can be significant sources of VOCs and other contaminants. This should be considered during risk assessments. 47 Scrubs and Equipment Scrubs, hair covers, and shoe covers should be non-shedding, non-static, and color -stable under conditions of washing, drying, and sterilization. 48 Detergents Laundry detergents used for cleaning scrubs and equipment should not release VOCs.

100. Third-Hand Smoke Operators as Sources of VOCs Scrubs, hair covers, and shoe covers should be non-shedding, non-static, and color -stable under conditions of washing, drying, and sterilization. Laundry detergents used for cleaning scrubs and equipment should not release VOCs.

101. 49 Photocopiers and Printers Photocopiers and printers (laser or inkjet) emit VOCs, ozone, solvents, and toner dust. They should not be placed in the ART suite . If unavoidable, they should be housed in sealed cabinets under continuous negative pressure . 50 Computers Desktop computers emit VOCs and formaldehyde, particularly as they age. Use a minimum number of computers, switch them off when not in use, and off-gas new computers by running them for 10 days outside the ART suite. Tablets and smartphones emit fewer VOCs. Risk Assessment for Equipment: Laboratories should conduct risk assessments regarding the use of computing and printing equipment in cleanroom environments to minimize VOC contamination.

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