1. 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

2. Contents
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Importance of air quality in IVF
Regulatory bodies: what they say
How we implemented AQC
What we achieved

3. ANDROFERT
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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4. ANDROFERT
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
Infertile IVF
couples,
observational
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
Infertile IVF
couples,
observational
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
Infertile IVF
couples,
observational
IVF lab redesigned to
meet the EU directive
Implantation and pregnancy rates
increased after the move into the
cleanroom
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5. 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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6. 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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7. What regulatory agencies ask for
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8. 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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9. 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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10. 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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11. ANDROFERT
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12. 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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14. 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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15. 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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16. 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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17. WHAT PATIENTS USUALLY
SEE IS…

18. …AND NOT WHAT IS BEHIND THE DOORS!

19. HOW WE IMPLEMENTED AQC
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20. 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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21. AIR-HANDLING VENTILATION SYSTEM
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22. ANDROFERT
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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

23. ANDROFERT
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24. ANDROFERT
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2013 DECEMBER
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25. ANDROFERT
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26. ANDROFERT
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27. ANDROFERT
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28. ANDROFERT
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29. ANDROFERT
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30. ANDROFERT
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31. ANDROFERT
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32. HOW WE MONITOR
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33. 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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34. ANDROFERT
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35. Esteves & Bento. RBM Online 2013
ü APPROVED
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36. 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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37. 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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38. WHAT WE ACHIEVED
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39. 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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40. On average, an extra embryo for
transfer or cryopreservation
Air Quality Control & IVF Results
2,315 patients; 14,660 embryos
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41. Cumulative live birth rates in IVF
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
ANDROFERT
332/822 63/239 17/49

42. 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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43. 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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44. ThankYou
obrigado