implementation of air quality control in the ivf laboratory and other critical areas

Sandro C. Esteves, MD, PhD Director, ANDROFERT

Andrology & Human Reproduction Clinic Campinas, BRAZIL

Implementation of air quality control in the IVF lab and

other critical areas

TQM Summit, CHINA 2014

Contents

ANDROFERT androfert.com.br

ANDROLOGY AND HUMAN REPRODUCTION CLINIC REFERRAL CENTER FOR MALE REPRODUCTION PROF. DR. SANDRO ESTEVES 2013 OCTOBER






ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION SC ESTEVES, 2 2014 March

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Importance of air quality in IVF

Regulatory bodies: what they say

How we implemented AQC

What we achieved

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IMPORTANCE OF AIR QUALITY IN IVF Evidence from Animal/Experimental Studies

Study Population Method Outcome

Little and Mirkes, 1990

In vitro cultured rat embryos

Protein and DNA analysis High levels of acrolein associated with embryotoxicity

Cohen et al, 1997

Analytical measurement procedure

Air sampling and VOC determination in human IVF labs

Higher levels of VOC in HEPA-filtered lab ambient air and incubators compared with outside ambient air

Hall et al, 1998

Analytical measurement procedure and bioassay

Air sampling and VOC determination in human IVF laboratories; acrolein bioassay using 2-cell mouse embryos

Increased levels of VOC in ambient air. Reduction in aldehyde levels by air filtration using carbon-activated and permanganate. In vitro mouse embryo development, implantation and post-implantation outcome inversely correlated with acrolein levels

Worrillow et al, 2002

Analytical measurement procedure

Design of a high velocity air control system and laboratory with VOC air filtration

IVF laboratory and procedure rooms qualified as class 100 areas. No VOCs found above >0.1 parts per billion



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IMPORTANCE OF AIR QUALITY IN IVF Evidence from Human Studies

Study Population Method Outcome

Mayer et al, 1999

RCT of infertile IVF couples

Embryo culture in incubators with and without VOC filtration

Higher PRs when embryos were cultured in incubators with VOC filters

Racowsky et al, 1999

Infertile IVF couples, observational

IVF and embryo culture in labs and incubators with and without VOC filtration

Reduction in miscarriage in cycles performed in labs and incubators with carbon-activated filters

Boone et al, 1999


Construction of cleanrooms for IVF activities

Reduction in air particles associated with an increase in the number of high-quality embryos

Worrillow et al, 2002


IVF in clean rooms with VOC filtration. Outside and inside ambient air monitored for 2 years

Seasonal correlation between temperature/humidity of outside ambient air serving the IVF air system and VOC levels with impact on IR

Knaggs et al, 2007


IVF lab redesigned to meet the EU directive

Implantation and pregnancy rates increased after the move into the cleanroom



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IMPORTANCE OF AIR QUALITY IN IVF Evidence from Observational Human Studies

25.0%

32.7%

14.1%

43.1%

Miscarriage rate

CPR

General IVF Population (N=468)

Cleanroon IVF lab Standard IVF lab

CPR Miscarriage

rate

36.9%

23.0%

47.1%

15.0%

Severe Male Factor Infertility (N=399)

Standard IVF lab Cleanroom IVF lab

P=0.01

P=0.03

Esteves et al. Fertil Steril 2004 Esteves et al. Fertil Steril 2006



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Importance of Air quality in IVF

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ü High levels of VOC in unfiltered IVF lab air

ü Efficient particle and VOC removal by air filtration ü Mouse embryo development inversely correlated with

VOC ü Better outcome (PR, IR, miscarriage) in IVF cycles

performed laboratories with air particle and VOC filtration



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What regulatory agencies ask for

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androfert.com.br ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION SC ESTEVES, 7 2014 March

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Region (directive)

United States (FDA 21 CFR 1271.195)

European Union (EU directive 2004/23/EC; 2006/86/EC)

Brazil (Anvisa RDC33/2006; RDC23/2011)

Particle filtration

Process-based; specifications not defined

Equivalent to GMP Grade A air quality in the critical areas with a background environment at least equivalent to Grade D (exceptions apply)

At least equivalent to ISO class 5 (NBR/ISO 14644–1) in the critical areas

Microbial contamination

Process-based; specifications not defined

Microbial colony counts equivalent to those of Grade A as defined in the current GMC guide with a background environment at least equivalent to Grade D

Microbiological monitoring required; specifications not defined

Volatile organic compounds filtration

Not required

Not required

Ventilation systems should be equipped with filters imbedded with activated charcoal



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Social responsibility to protect public health from exposure

to harm, when scientific investigation has found a

plausible risk even though a scientific consensus had not

been reached

PRECAUTIONARY PRINCIPLE

*Convention on Biological Diversity, UN 1992; Ratified by 192 countries and the EU, except USA, Andorra and South Sudan



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CONTAMINATION Any undesirable material or substance

that may affect the final product quality

1. Airborne particles Residues of cleaning agents and clothing, skin shedding; temporary

suspension; 1< µm <100

2. Microorganisms Viruses, spores, bacteria; constant suspension in air; size <1 up to 8µm

Contaminants are generated by laboratory personnel, processes,

furniture and equipment



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CONTAMINATION

3. Indoor Volatile Organic Compounds

Chemical compounds that evaporate and react with indoor ozone;

Result in submicronic particles and harmful by-products (toluene, benzene, alcohols,

acetone, refrigerating gases, aldehydes) Cigarette smoke, automobile exhaust,

detergents, lubricants, dyes, furniture, rubber, plastics, resins, synthetic fibers, nylon,

particle-board, plywood



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REPRODUCTIVE LABORATORIES

Indoor air

Personnel

Material and Equipment

Cell, tissue and fluids

0

1000

2000

3000

Outside Air Indoor IVF

Lab Air

533

2769 5-fold increase in VOC

Anderson et al. 1997



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HOW TO PROTECT GAMETES AND EMBRYOS FROM CONTAMINATION

Bacteria and other contaminants can attach themselves to particles, a decrease in particles equates to an increase in air quality Pre-Filters: >10µ

Fine dust filters: 1<10µ HEPA filters: 0,3µ = 99,97%

Removal of air particulates by forced air movement using positive air pressurization

through a series of filters with increased efficiency



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VOCs are 100–1000x smaller than the effective pore size of HEPA filters

Spaces between the carbon particles contain a cloud of

delocalized electrons that act as electronic glue to chemical

contaminants

Alcohols and ketones are oxidized and thereby

detoxified by potassium permanganate

HOW TO PROTECT GAMETES AND EMBRYOS FROM CONTAMINATION

Removal of VOCs by forced air movement using positive air pressurization through a

series of filters containing activated carbon and potassium permanganate



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WHAT PATIENTS USUALLY SEE IS…

…AND NOT WHAT IS BEHIND THE DOORS!

HOW WE IMPLEMENTED AQC

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LAYOUT AND CONSTRUCTION

In situ wet construction Low off-gassing materials

Surfaces and Coatings Lighting fixtures

Flush-mounted and sealed Coved junctures Stainless steel and aluminium

Furniture, doors, windows, air vents, workstations



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AIR-HANDLING VENTILATION SYSTEM



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Particle size/m3 of air: ISO 14.644-1 0.3 µM 0.5 µM 1 µM 5 µM

ISO 1 ISO 2 10 4 ISO 3 102 35 8 ISO 4 1,020 352 83

IVF lab ISO 5 10,200 3,520 832 29 ISO 6 102,000 35,200 8,320 293

OR ISO 7 352,000 83,200 2,930 Transfer ISO 8 3,520,000 832,000 29,300

ISO 9 35,200,000 8,320,000 293,000

CLEAN ROOM CLASSIFICATION



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ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION SC ESTEVES, 24 2013 DECEMBER

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HOW WE MONITOR



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TEST EQUIPMENT

Air volume flow rate Termoanemometer

Air exchange rate Termoanemometer

Air and room pressure differential

Microanemometer and balometer

HEPA-filter integrity leak Aerosol generator

Airborne particle counts Electronic particle counter

Recovery performance Smoke generator

Lighting level Luxmeter

Noise level Decibelmeter Temperature and humidity

Thermometer and hygrometer

Third-part certification company (CCL, Brazil); semi-annually IEST 006.2 standards



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Esteves & Bento. RBM Online 2013

ü APPROVED



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Settle plates Mold and blood-agar Petri dishes

Surface sampling Swabs and contact plates

Five-finger glove printing

Incubators, workstations, air, floor, wall

Limits Colony Forming Unites (CFU)

Settle plates (90mm

diameter) CFU/4 hours

Contact plates

(55mm diameter) CFU/plate

Glove print CFU/glove

<3 <3 <3

Microbiological Monitoring



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Volatile Organic Compounds Monitoring

Activated-carbon life-time estimates Carbon tetrachloride activity method

Active air sampling on Tenax TA sorbent and 2,4 dinitrophenyl hydrazine

Summa canisters Thermal desorption Gas chromatography (Limits < 2 mcg/m3)



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WHAT WE ACHIEVED

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Air Quality Control & IVF Results 2,315 patients; 14,660 embryos

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

Before After 2002-2004

After 2005-2007

After 2008-2010

TQE D3 (%) Miscarriage (%) LBR fresh (%)



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On average, an extra embryo for transfer or cryopreservation

Air Quality Control & IVF Results 2,315 patients; 14,660 embryos



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Cumulative live birth rates in IVF

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40.4% 48.0%

Esteves, 41 ANDROFERT, Referral Center for Male Reproduction

ET #3 (FET) 49

ET #2 (FET) 239

ET #1 (fresh) 822

50.5% +18.8%

+25.0% Female Age ≤38

AN

DR

OF

ER

T

332/822 63/239 17/49

Conclusions (1) Air quality (particulate matter and VOC) is associated

with in vitro embryo development in animal and human studies

Regulatory agencies have issued Directives with specific

requirements for air quality in IVF labs

We set up an IVF facility (embryology and andrology lab, operating room, embryo transfer room) with clean room technology, and demonstrated how feasible it is to work in such environments



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Conclusions (2)

Our ART program have performed better in Clean Room environments for over a 7-year period

Costs of Clean Room Technology:

Implementation: USD 150,000.00 Operation: USD 15,000.00/year

filters, clean room utensils/material, maintenance, certification Additional cost per cycle: ~USD 130



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implementation of air quality control in the ivf laboratory and other critical areas

Health & Medicine