Air quality in the clinical embryology laboratory: a mini-review

Abstract

The scope of the clinical embryology laboratory has expanded over recent years. It now includes conventional in vitro fertilization (IVF) techniques and complex and time-demanding procedures like blastocyst culture, processing of surgically retrieved sperm, and trophectoderm biopsy for preimplantation genetic testing. These procedures require a stable culture environment in which ambient air quality might play a critical role. The existing data indicate that both particulate matter and chemical pollution adversely affect IVF results, with low levels for better outcomes. As a result, IVF clinics have invested in air cleaning technologies with variable efficiency to remove particulates and volatile organic compounds. However, specific regulatory frameworks mandating air quality control are limited, as are evidence-based guidelines for the best air quality control practices in the embryology laboratory. In this review, we describe the principles and existing solutions for improving air quality and summarize the clinical evidence concerning air quality control in the embryology laboratory. In addition, we discuss the gaps in knowledge that could guide future research to improve clinical outcomes.

Full text

Ther Adv Reprod Health
2021, Vol. 15: 1–11
DOI: 10.1177/
2633494121990684
© The Author(s), 2021.
Article reuse guidelines:
sagepub.com/journals-
permissions
Therapeutic Advances in Reproductive Health
journals.sagepub.com/home/reh 1
Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License
(https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission
provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage).
Background
The number of couples facing infertility has
increased steadily, many of whom will ultimately
need medically assisted reproductive (MAR)
treatment. Global data report over 8 million in
vitro fertilization (IVF) babies born in the last
40 years, and in the UK, IVF babies account for
about 3% of all babies born in 2016.1,2
Furthermore, in the last decades due to the social
and legal equality for same-sex couples, MAR
treatment are increasingly applied for those cou-
ples as well as single women/men and transgender
couples. Consequently, increasing numbers of
homosexual are seeking help to assisted reproduc-
tive technology (ART) to achieve parenthood in
countries where it is allowed.3 IVF is a high-com-
plexity multi-step procedure, which has markedly
evolved over the last decades.4 Human embryo-
genesis involves a coordinated cascade of bio-
chemical and molecular intracellular signaling
events between gametes that results in the devel-
opment of viable embryos capable of implantation
and establishment of viable pregnancies to term.
Indeed, the extended in vitro culture of human
embryos constitutes one of the most challenging
applications of cell culture. This process demands
a more critical growth environment as gametes
and embryos are especially sensitive cells types,
largely unprotected as they lack epithelial surfaces,
immunological defenses, detoxifying mechanisms,
thus being vulnerable to environmental influ-
ences. Studies that have examined environmental
and airborne pathogens have indicated that both
ambient air pollution as well as laboratory air
quality (AQ) may play a significant role on embry-
ogenesis, implantation, and conception of MAR
treatments.5,6 Thus, efforts have been devoted to
optimizing the embryology laboratory environ-
ment to mitigate the possible adverse effect of
ambient air on IVF outcomes. Our review aims to
(1) describe the principles and existing solutions
for improving laboratory air quality, (2) summa-
rize the existing evidence concerning AQ control
in the embryology laboratory, and (3) highlight
Air quality in the clinical embryology
laboratory: a mini-review
Romualdo Sciorio , Erika Rapalini and Sandro C. Esteves
Abstract: The scope of the clinical embryology laboratory has expanded over recent years.
It now includes conventional in vitro fertilization (IVF) techniques and complex and time-
demanding procedures like blastocyst culture, processing of surgically retrieved sperm, and
trophectoderm biopsy for preimplantation genetic testing. These procedures require a stable
culture environment in which ambient air quality might play a critical role. The existing data
indicate that both particulate matter and chemical pollution adversely affect IVF results,
with low levels for better outcomes. As a result, IVF clinics have invested in air cleaning
technologies with variable efficiency to remove particulates and volatile organic compounds.
However, specific regulatory frameworks mandating air quality control are limited, as
are evidence-based guidelines for the best air quality control practices in the embryology
laboratory. In this review, we describe the principles and existing solutions for improving air
quality and summarize the clinical evidence concerning air quality control in the embryology
laboratory. In addition, we discuss the gaps in knowledge that could guide future research to
improve clinical outcomes.
Keywords: air quality (AQ), assisted reproductive technology (ART), embryo culture, in vitro
fertilization (IVF), volatile organic compounds (VOCs)
Received: 26 June 2020; revised manuscript accepted: 7 January 2021.
Correspondence to:
Romualdo Sciorio
Edinburgh Assisted
Conception Programme,
EFREC, Royal Infirmary
of Edinburgh, 51 Little
France Crescent, Old
Dalkeith Road, Edinburgh
EH16 4SA, UK.
sciorioromualdo@hotmail.
com
Erika Rapalini
Ospedale Versilia,
Centro di Procreazione
medicalmente assistita,
Lido di Camaiore, Italy
Sandro C. Esteves
ANDROFERT, Andrology
and Human Reproduction
Clinic, Campinas, Brazil;
Division of Urology,
Department of Surgery,
University of Campinas
(UNICAMP), Campinas,
Brazil
Faculty of Health, Aarhus
University, Aarhus,
Denmark
990684REH0010.1177/2633494121990684Therapeutic Advances in Reproductive HealthR Sciorio, E Rapalini
review-article20212021
Review

2 journals.sagepub.com/home/reh
Therapeutic Advances in Reproductive Health 15
the main gaps in this area of knowledge that could
guide future research and improve ART clinical
outcomes.
What are the threats?
Particulate matter (PM) is a mixture of microscopic
solids and liquid droplets measuring from 1 to 100
microns, in temporary suspension in air. The
embryology laboratory may be served by outdoor
air, whose quality is influenced by many factors,
including construction, vehicle traffic and exhaust,
industrial and commercial emissions, waste man-
agement, and seasonal pollutants, to cite a few.
Microorganisms, like viruses, spores, and bacteria,
measure from < 1 to up 8 microns, and are present
on all inanimate surfaces and in air suspension, cre-
ating potentially sources of contamination in the
embryology laboratory. They can adhere to PM
and contaminate surfaces when the particles set-
tle. Volatile organic compounds (VOCs) are any
organic (carbon-containing) solid or liquid com-
pound that evaporates at room temperature.
They react with indoor ozone and create submi-
cronic particles and harmful by-products, some of
which have toxic properties and are potentially
mutagenic.7 VOCs are generated from a variety of
materials,8 the list is long and includes construction
materials, such as wood furniture,9 polyvinyl chlo-
ride flooring materials,10 adhesives, and paints,11 all
of which release formaldehyde or aldehydes.
Cosmetics like perfumes and aftershaves also
release VOCs due to evaporation of their solvent,
which is typically alcohol-based. Autoclaved mate-
rials release VOCs when packs are opened for use.
Also, laboratory plasticware, which is made from a
variety of plastics like polyethylene, polystyrene,
polycarbonate, polypropylene, and acrylic, release
VOCs. These compounds can also be found in
CO2 gas cylinders, insulation used in air handling
systems, and refrigerant gases.5,8,10 Similarly, clean-
ing products can be a source of VOCs (e.g. vinyl
floor liquid wax and ammonia-based products,
glass cleaners, and aerosols that contain butane or
isobutane as propellants).12 Although low-VOCs
disinfectant, specifically designed to use in IVF
laboratories, have been introduced, many laborato-
ries still use ethanol-based products, which despite
its effect against viruses and bacteria, release VOCs.
Finally, mold growth produce carbon dioxide,
water and VOC. VOCs and other small inorganic
gaseous molecules, including nitrous oxide, sulfur
dioxide, and heavy metals, seem to be detrimental
to embryo development.13,14 Furthermore, VOCs
can harm sperm quality by attaching to the
DNA, causing fragmentation and altering cell
replication.15
Detecting VOCs in the embryology
laboratory
In the clinical embryology laboratory, VOC levels
are generally measured using portable direct read-
ing instruments using VOC probes. Short- and
long-term sampling (hours or days) can be utilized
to provide a snapshot or average exposure over
time, respectively. These instruments determine
the total VOC concentration (TVOC), that is, the
total concentration of all VOCs in a defined meas-
uring range, using the photo-ionization detection
(PID) method. The measurements are expressed
in parts per million (ppm) or parts per billion
(ppb), depending on the instrument’s type and
detection limits. Although the PID method has the
advantage of obtaining results immediately, it does
not identify the VOC type or quantify them sepa-
rately. The latter will require active sampling on
TenaxTA® Sorbent, thermal desorption, and gas
chromatography using mass spectrometry, which
provides detailed information about non-polar and
weakly polar VOCs. Aldehydes will need different
measurement methods (eg., 2.4-dinitrophenylhy-
drazine impregnated silica tubes or cartridges with
subsequent solvent desorption, clean-up and liq-
uid chromatographic analysis). Therefore, the
VOC assessment in indoor air is highly dependent
on how this evaluation is performed. All available
methods are selective in what they can measure,
and there is no device capable of measuring all
VOCs. Also, the type of instrument chosen will
determine the sensitivity of the measurements.14
Importantly, the portable devices that measure
TVOCs in ppm might not be able to detect poten-
tially genotoxic, or mutagenic VOCs due to their
low detection limits.16 In the IVF laboratory, it
would be more appropriate to perform VOC meas-
urement with instruments providing readings in
ppb. The results might reflect better the indoor air
quality concerning the potential genotoxicity of the
biochemical interaction between gametes/embryos
and contaminants. In one of the author’s (S.C.E.)
facility, a portable VOC meter in ppb is turned on
during operational hours; 100 ppb is set as the
alarm level above which critical activities such as
incubator openings and gamete/embryo manipula-
tion should be avoided. Threshold values above

R Sciorio, E Rapalini et al.
journals.sagepub.com/home/reh 3
which an adverse effect of VOCs is to be expected
in the context of human cell culture are yet to be
established. Studies have reported that increased
levels of VOCs were related with a statistically sig-
nificant decrease in clinical pregnancy rates, but it
remains mostly unknown the specific thresholds
according to the type of VOC, except for alde-
hydes, which should be kept below the detection
limit of 80–100 ppb.8,17–19 Currently, it has been
generally recommended that IVF laboratories
maintain total VOC levels below 400 to 800 ppb.20
However, a study by Worrilow and colleagues,17
including 8 years of clinical outcomes, and evaluat-
ing the dynamic levels of VOCs and viable and
nonviable particulates within the ambient air of the
IVF laboratory, indicated that VOC levels far
below 100 ppb affected preimplantation embryo-
genesis negatively.
Solutions to improve air quality
The principles of AQ control in the embryology
laboratory involves four main aspects, namely:
(1) air pressure differential, (2) turbulent air, (3)
high-efficiency particulate air (HEPA) filtration,
and (4) VOCs filtration.21 Positive pressure cre-
ates an air pressure differential between adjacent
rooms, thus minimizing retention as both particu-
late matter in air suspension and VOCs are car-
ried away, whereas the newly filtered air dilutes
the remaining particles and VOCs. Air pressuri-
zation also creates turbulent air, which washes
out “dead” air in critical spots like those under
workstations, microscopes, and other equipment.
Similarly, the introduction of particles and VOCs
from the outside air is avoided by the forced air
movement, which uses positive air pressurization
through filters. As mentioned above, microorgan-
isms can adhere to PM; thus, a decrease in the
number of particles in air suspension equates to a
decrease of contamination by pathogens. Removal
of air particles is commonly achieved with the use
of HEPA filters. These filters are designed to
remove 99.97% of particles greater than or equal
to 0.3 microns. However, due to their physical
characteristics, HEPA filters remove not only
particles measuring 0.3 microns or higher (by
sieving or impaction) but also smaller particles
the size of viruses (eg., SARS-CoV-2; ~0.1
microns) by diffusion and interception methods.22
VOCs removal is also critical for improving the
AQ in the embryology laboratory. Typically,
VOCs are removed by sorption filtration (mass
transfer from air to adsorbent) using chemical fil-
ters, typically manufactured as a mesh embedded
with activated carbon, potassium permanganate,
activated alumina, and silica gels.23 The space
between the carbon particles contains delocal-
ized electrons that act as electronic glue, thus
inducing the chemical contaminants to attach to
the carbon.24 Alcohols and ketones, which can-
not be removed by carbons filters, are detoxified
with the use of potassium permanganate.14
Alternatively, VOCs can be removed using ultra-
violet photocatalytic oxidation (UVPCO), gen-
erally combined with the use of carbon filters.25
UVPCO uses the energy of UV lights absorbed by
a semiconductor metal oxide (eg., titanium oxide)
to produce reactive species which adsorb VOCs.
The photo-oxidation of VOCs causes the produc-
tion of CO2, water, and partially oxidized by-
products. Volatile by-products can be released as
secondary pollutants, whereas the filters adsorb
non-volatile by-products. Thus, the real effective-
ness of UVPCO filters is still debatable and not
wholly accepted by the scientific community.26,27
Many air cleaning solutions are commercially
available with variable efficiency to control air
contamination. One option consists of portable
filtration systems, first proposed by Cohen and
colleagues.12 This system employs stand-alone
VOCs filtration units equipped with VOCs and
HEPA filters; some units may also have UVPCO
(Figure 1). Data on their efficacy are minimal,
and it is still premature to make a firm conclusion
on the actual effectiveness of these portable
units.24,25 Another option is the use of centralized
air filtration systems, in which a mix of outside
and recirculated indoor air is pressurized and fil-
tered through a series of dust, VOCs, and termi-
nal HEPA filters (Figure 2). Typically, the
amount of outside air and recirculated air in IVF
laboratory cleanrooms is 20%–25% and 75%–
80%, respectively. This balance is important as
recirculated air is stable and has already been fil-
tered, thus securing the optimal life for the expen-
sive HEPA filters and reducing the running costs
and energy waste. On the other hand, VOC can
accumulate in the indoor environment, and fresh
outside air provides a way of both diluting VOCs
and reducing the risk of cross-contamination
between spaces. The filtered air is distributed to
the embryology laboratory and adjacent rooms
via ducts. The choice of the optimal filtration sys-
tem should be based on a risk management analy-
sis. This analysis should include the design,

4 journals.sagepub.com/home/reh
Therapeutic Advances in Reproductive Health 15
Figure 1. Illustration depicting a portable four-stage free-standing air filtration system. Reprinted from: Sadir
and colleagues,22 with permission from Taylor & Francis.
Figure 2. Schematic representation of cleanrooms (embryology suite, operating theater, and embryo transfer
suite). Airflow patterns and filtration units are also depicted. The air-handling ventilation unit is located in a
separate room. An external rooftop subunit draws outside air that goes through coarse and activated carbon
prefilters before entering into the main unit. The main ventilation unit pulls prefiltered outside air and the
cleanrooms’ return air through coarse filters, past a 16-unit potassium permanganate-impregnated pelletized
coconut shell-based activated carbon filters and then through fine dust filters. Finally, filtered air enters the
cleanrooms through a set of high-efficiency particulate air (HEPA) filters. Floor- and ceiling-level vents in
the cleanrooms return air to the main ventilation unit to be remixed with the existing air. Differential positive
pressure is maintained between rooms. The embryology laboratory/anteroom is positive to the operating
room, which is positive to both the embryo-transfer room and the dressing room/hallways. Reprinted with
permission from Esteves and Bento.7

R Sciorio, E Rapalini et al.
journals.sagepub.com/home/reh 5
qualification, and operation of the embryology
laboratory.26 Critical aspects to be taken into
account include age and size of the laboratory,
outdoor ambient pollution, whether the facility is
old or new, which affects the VOCs levels, and
the existence of regulatory directives dictating the
air cleanliness to be achieved. While compact
cleaning solutions are less expensive and easy to
implement, they provide less control of air con-
tamination than centralized filtration systems due
to inherent technical limitations concerning their
capability to address the four principles of AQ
control discussed above. The available clinical
data—albeit limited—seem to favor the use of
cleanroom laboratories for AQ.27,28 However, it
remains to be determined if a cleanroom is neces-
sary as no randomized controlled trial has exam-
ined this issue. Also, it is not known if the practice
of using a cleanroom would be more useful in
specific situations, such as laboratories installed
in highly polluted areas. Attention to construc-
tion materials, equipment, furniture, and human
activity is also essential to reduce contamination.
The use of low off-gas materials is preferable, and
laboratory personnel should be trained on the
principles of air quality control, including the
function of airflows and airlocks, hygiene, dress
code, and the use of cleaning agents.5 Equally
important is the use of inline gas filtration systems
as compressed gases (eg., CO2) are known sources
of contaminants, including n-butane, acetalde-
hyde, isopropanol, freon, and benzene.29–33 The
use of modern incubators, properly designed for
the embryology laboratory, with built-in VOC
and HEPA filters (some with UV light) and thus
providing clean air in the embryo culture cham-
bers, may offer additional benefit. These incuba-
tors keep culture conditions, including pH,
temperature, and air quality steady through the
entire period of culture, and therefore, may offer
a valid option for AQC for laboratories that adopt
uninterrupted single step culture media.34
Monitoring
A risk management analysis is paramount to
determine what measures should be implemented
to mitigate the risks associated with chemical and
particulate contamination, as discussed above, as
many activities involving gametes and embryos
are performed in the laboratory environment.
Irrespective of the chosen filtration system, moni-
toring the AQ is also essential. The critical air
cleanliness elements to control periodically
includes (1) no. particles in air suspension, (2) air
pressure differential, (3) air exchanges per hour,
(4) total VOC in air, and (5) microbiological con-
trol. Microbiological contamination is a critical
element to be monitored and must be a part of
the routine quality control process, which is usu-
ally carried out periodically (every 3-6 months)
using air samples, sedimentation plates (e.g.
90 mm plates), and swabbing methods. The num-
ber of colony forming units is measured and the
type of microbial can be determined. Besides,
contamination control measures are of utmost
importance to mitigate the risk of microbial con-
tamination (e.g. use of gloves, masks, and cover-
alls). The heating, ventilation, and air conditioning
(HVAC) is an integral element of the air quality
system. It has to be properly installed, main-
tained, and cleaned. Air ducts, coils, drain pan,
grills, blower motor, air plenum, and air filters are
some of the parts that need to be decontaminated.
During HVAC cleaning, air pressurization should
be placed under negative pressure or vacuum to
prevent the spread of contaminants. The negative
pressure also helps to get rid of dust and fine par-
ticles, as well as loosened contaminants. After
mechanical cleaning, sanitizers are applied to
nonporous surfaces of HVAC systems to control
for microbial contamination. As for filter replace-
ment, it is essential to consider that filter satura-
tion depends on outside air quality and pollution
levels and on the strategies adopted by the embry-
ology laboratory to mitigate the introduction,
generation, and retention of particles and VOCs.
Thus, filter replacement should be guided by the
concurrent analysis of AQ rather than the manu-
facturer’s specifications. A detailed description of
how to perform AQ control in the embryology
laboratory can be found elsewhere.35–37
Summary evidence
In 1997, Cohen and colleagues12 investigated the
effect of chemical contamination on IVF outcomes
in different settings. In this study, a new IVF labo-
ratory was built above one of the busiest streets of
southern Italy, known for its high industrial emis-
sion and stagnant air. At the beginning of the clini-
cal activities, the authors reported a significant
drop in pregnancy rate. After the installation of a
pump to push air through a water-filtered gas bot-
tle before entering the incubator, the pregnancy
results were returned to average values. In another

6 journals.sagepub.com/home/reh
Therapeutic Advances in Reproductive Health 15
setting, the authors installed solid carbon and
potassium permanganate filters in the IVF labora-
tory to remove adhesive and paint smells and other
pollutants. Sixteen months after the installation,
they found an overall increased implantation rate
from 22% to 36% in a cohort of 1400 patients.
This study suggested that chemical air contamina-
tion might adversely affect in vitro embryo culture
and pregnancy outcomes, albeit the VOC levels
were not precisely evaluated before and after the
introduction of air filtration. The same group in a
subsequent trial investigated the effect of acrolein
on mouse embryo development.14 Acrolein belongs
to the group of aldehydes, and is found in the air,
probably as a result of industrial activity. The com-
pound has also been detected in tobacco smoke.38
The authors found that the mouse embryos
growth was significantly affected after acrolein was
added at different concentrations to the culture
environment.14 The physiological effect was noted
at concentrations in the low ppm range. Although
this is not a typical condition in ART, it could be,
however, speculated that acrolein and other alde-
hydes might be associated with reduced growth of
mammalian embryos.39 Other studies in the late
1990s provided insights into the compounds and
factors that impact AQ. Schimmel and colleagues40
showed that both incubators and compressed
medical gasses are sources of VOCs, as well as ster-
ile Petri dishes, incubators, cleaning supplies,
monitors, microscopes, and even furniture, all of
which contribute to the emission of VOCs.13 A
study published by Nijs and colleagues used the
human sperm survival test to identify potential
reprotoxic products and consumables used in ART
procedures. They analyzed several products cus-
tomarily used in the IVF laboratory, including sur-
gical gloves, ovum pickup needle, type of embryo
transfer catheter, and also sterile Pasteur pipette
and culture Petri dish, and found that 13 of 36
products analyzed were potentially toxic. The
authors speculated that these products might
release chemical compounds toxic to sperm.41
Another critical aspect to consider concerning
chemical contamination to human embryo culture
is the use of mineral oil as a protective overlay dur-
ing human embryo culture. On the one hand, an
oil overlay could be a barrier to water-soluble
VOCs such as ethanol, acetone, and formalde-
hyde. On the other hand, benzene, isobutylene,
and styrene are oil-soluble, highlighting the impor-
tance of controlling VOC sources in the IVF lab.
Added to this, mineral oil is a petroleum-derived
product that can vary widely in quality.
Commercially available oil might contain aromatic
and unsaturated hydrocarbons that are susceptible
to peroxidation and free radical formation,42 par-
ticularly during storage or culture conditions.43
Lately, with the widespread use of the single-step
medium in human in vitro embryo culture, the
mineral oil might be exposed to a longer incuba-
tion time at 37°C up to 7 days, thus increasing the
risk of peroxidation and generation of toxic com-
pounds that could be directly transferred to the
embryo.44 In a 1997 retrospective cohort study
involving infertile couples undergoing IVF, Boone
and colleagues31 observed that the construction of
class 100 cleanroom for air particulates improved
air quality and increased the number of high-qual-
ity embryos available for transfer, ultimately
increasing clinical pregnancy rates. Later in 2013,
Esteves and Bento5 also reported their results after
the construction of a new IVF facility that used a
centralized air ventilation system to supply filtered
air in terms of particles and VOCs to the IVF labo-
ratory, operating room and embryo transfer room.
The new filtration system used forced air move-
ment through a series of pre-filters, 16 beds of acti-
vated carbon mixed with potassium permanganate
and silica, and HEPA filters before the air supplied
the critical areas. The authors reported that during
the study period, few changes other than the envi-
ronmental conditions were made. They measured
air particles and VOC levels before and after the
installation of the new system and noticed a sharp
decrease in both contaminants. In parallel, they
showed an increased live birth rate (35.6% vs
25.8%, P = .02) and decreased miscarriage rate
(20.0% vs 28.7%; P = .04) compared to their
results before the implementation of AQ.5 In 2015,
Munch and colleagues45 assessed the fertilization,
cleavage, blastocyst formation, clinical pregnancy,
and live birth rates in the presence and absence of
carbon filtration. This retrospective cohort
included a total of 524 fresh cycles. The authors
found that fertilization, cleavage, and blastocyst
formation rates were significantly reduced in fresh
IVF when, by mistake, the carbon filters were not
replaced. They also noticed that these metrics
returned to normal ranges when new carbon filters
were installed, thus suggesting that the lack of car-
bon air filtration adversely affected embryo devel-
opment, in particular, around the time of
fertilization, when the oocytes are very sensitive to
air quality changes.45 However, VOC levels were
not reported before and after filters’ replacement,
and it remains speculative if indeed VOC levels
were high in the absence of carbon filtration. In

R Sciorio, E Rapalini et al.
journals.sagepub.com/home/reh 7
another study, Heitmann and colleagues32 assessed
IVF pregnancy outcomes before and after renovat-
ing their IVF facility. The new facility included a
dedicated centralized air filtration system for parti-
cles and VOCs. The authors emphasized that no
changes occurred in the personnel, laboratory
equipment, or protocols during this period.
Overall, the total VOC (819.4 g/m3 vs 32.0 ug/m3)
and aldehyde (13.7 µg/m3 vs 5.2 µg/m3) concentra-
tions in the IVF laboratory decreased after the
installation of the new AQ system. The decrease in
VOC levels were associated with increased rates of
implantation (24.3% vs 32.4%, P < .01), as well as
clinical pregnancy (40.8% vs 50.2%, P = .01) and
live birth (31.8% to 39.3%, P = .03). Table 1 sum-
marizes the relevant clinical studies on AQ and
IVF outcomes published to date. These studies
collectively suggest that laboratory air filtration
might improve both embryonic and pregnancy
outcomes of infertile couples undergoing ART.
However, it is worth noting that the existing stud-
ies are mainly retrospective in nature and lack
proper control groups. Moreover, the implemen-
tation of other relevant changes rather than air
filtration alone could have positively impacted
IVF outcomes. Thus, a cause-effect relationship
remains to be demonstrated in prospective con-
trolled trials.
Gaps in knowledge and future directions
Due to the increasing evidence suggesting an asso-
ciation between laboratory air quality and ART
outcomes, regulatory bodies issued cleanroom
specifications.48,49 While these directives are
intended to safeguard public health in line with the
precautionary principle, they differ in the specifica-
tions on how AQ control has to be handled.5 For
instance, the EU directive focuses on particulate
air only, whereas the Brazilian directive tackles
both particles and VOCs. Along the same lines,
VOC thresholds for embryology laboratories are
lacking, as are specific practice guidelines on good
embryology laboratory practices concerning AQ
control. Nevertheless, a recent document dis-
cussed the relevant aspects concerning AQ control
in the embryology laboratory and provided practi-
cal recommendations based on expert judgment to
guide laboratory design, qualification, and opera-
tion concerning AQ control.20 Nevertheless, level 1
evidence is still lacking to support any recommen-
dation concerning the minimum AQ requirements
for optimal human gamete manipulation and
embryo culture. Indeed, several gaps in knowledge
exist in this area. Notably, it is challenging to per-
form well-designed studies on the effect of air
quality and environmental pollutants on embryo
development. First, there are many chemical com-
pounds partitioned in the air. Second, the VOC
mass transfer from air to water/culture phase of the
cultured cells and embryos is difficult to model.
Nowadays, culture systems are not biphasic due to
the effect of the commonly used oil interphase.
Thus, all gaseous interactions occur through min-
eral oil. Third, the relative solubility of a chemical
compound in oil is described by its oil–water parti-
tion coefficient. Partitioning into mineral oil or
water/culture medium is not the same for different
classes of compounds. Compounds with a high
partition coefficient are hydrophobic (e.g. ben-
zene, styrene) and achieve a much higher concen-
tration in the oil phase than in the aqueous phase
at equilibrium. By contrast, hydrophilic com-
pounds (e.g. ethanol, acrolein) accumulate prefer-
entially into the aqueous phase. Future studies
should focus on the objective determination of the
most relevant chemical contaminants in the IVF
lab, and their thresholds. In this sense, triphasic
models would be essential to study the effects of
chemical contamination on embryo development.
Equally important would be to investigate (e.g.
using well-designed pragmatic clinical trials) the
effectiveness of different air filtration approaches
(eg., cleanroom vs portable filtration systems), and
whether modern “sealed” incubators equipped
with VOC and HEPA filters could compensate the
lack of stringent AQ control. The results of these
studies will be invaluable to elaborate evidence-
based practice guidelines for AQ control in embry-
ology laboratories.
Conclusion
The association between the environment, preim-
plantation toxicology, and successful embryogen-
esis demands a comprehensive evaluation of the
main variables that contribute to air contamination
in the embryology laboratory. Particulate matter
and VOCs are the primary pollutants to be con-
trolled for in the embryology laboratory as they
might adversely affect embryogenesis, implanta-
tion, and conception in ART cycles. Embryology
laboratories that are willing to adopt strict air qual-
ity control should be constructed and used to min-
imize the introduction, retention, and generation
of particles and VOCs, in which temperature,
humidity, and pressure are continuously controlled
and monitored. Attention should also be paid to

8 journals.sagepub.com/home/reh
Therapeutic Advances in Reproductive Health 15
Table 1. Evidence summarya concerning the impact of air quality control in the embryology laboratory on the outcomes of assisted reproductive technology cycles.
Study Study design Study population Air quality control Outcome
Schimmel and
colleagues40
Descriptive qualitative
study
None Air sampling and VOCs in human
IVF laboratories
Higher levels of VOC found in CO2 tanks and incubators compared to outside air.
Air filtration using carbon activated and potassium permanganate reduced VOC levels
Hall and colleagues14 Descriptive qualitative
and observational
cohort studies
In vitro mouse
embryos
Air sampling and VOCs in human
IVF laboratories and Acrolein
bioassay using mouse embryos
Increased levels of VOC observed in ambient air of human IVF laboratories.
Reduction in aldehyde levels by air filtration using carbon-activated and
permanganate.
In vitro mouse embryo development, implantation, and post-implantation
development inversely correlated with acrolein concentration
Boone and colleagues31 Observational study 275 infertile couples
undergoing IVF;
fresh ET
Centralized system (Class 100
cleanroom) for particle filtration.
Air particles decreased (P < .001) after implementation of centralized air filtration.
Reduction in air particles was associated with an increase in the number of high-
quality embryos (62% vs. 71, P < .001) and CPR per transfer (59% vs. 35%, P = .003).
Khoudja and
colleagues52
Descriptive qualitative
and observational study
1403 infertile
couples undergoing
IVF/ICSI cycles;
fresh ET
Combination of centralized and
portable system to filter particles
and VOCs
VOC levels decreased and overall air quality improved after installation of
centralized VOC air filtration system.
Higher fertilization (83.7% vs. 70.1%, P < .001) blastocyst rate (51.1% vs. 41.7%,
P < .001), IR (34.4% vs. 26.4%, P = .001), and CPR (54.6% vs. 40.6%, P = .001) were
observed after the incorporation of strict air quality control.
Esteves and Bento5Descriptive qualitative
and observational study
2060 ICSI cycles;
fresh ET
Centralized system for particles
(ISO 5 cleanroom) and VOC
filtration (new facility) compared
with portable air filtration system
(old facility)
Higher rates of high-quality embryos and live birth rates (35.6% vs. 25.8%, P = .02),
and lower miscarriage rates (28.7% vs. 20.0%, P = .04) in the new facility than in the
old facility.
Munch and colleagues45 Observational study 524 fresh and 156
cryopreserved IVF/
ICSI cycles
VOC and HEPA air filtration (not
specified if centralized or portable)
Embryonic (cleavage and blastocyst rates rates) and pregnancy (IR, CPR, LBR)
outcomes decreased significantly (P < .05) in fresh cycles carried out in the absence
of VOC air filtration and increased (P < .05) after installation of VOC filters.
Heitmann and
colleagues32
Combination of
descriptive qualitative
and observational study
820 IVF/ICSI cycles;
fresh ET
Centralized system for particle
and VOC filtration (new facility)
compared with portable air
filtration system (old facility)
Lower VOC levels (Total VOC 819.4 μm3 vs. 32 ug/m3, and aldehyde 13.69 ug/m3 vs.
5.2 ug/m3), and higher IR (32.4% vs. 24.3%; P < .01) and LBR (39.3% vs. 31.8%, P = .03)
in the new facility than in the old facility.
Agarwal and
colleagues53
Combination of
descriptive qualitative
and observational study
1036 IVF/ICSI cycles;
fresh ET
Portable air filtration system for
particles and VOCs
Lower VOC levels (P < .0001), higher No. blastocysts (6.3 ± 0.8 vs. 4.5 ± 0.4; P = .04),
and higher IR (42% vs. 31%, P = .001) as well as LBR (31% vs. 23%, P = .009) after
incorporation of air filtration.
CPR: clinical pregnancy rate; ET: embryo transfer; HEPA: high-efficiency particulate air; IR: implantation rate; ISO: international organization for standardization; IVF: in vitro fertilization; ICSI:
intracytoplasmic sperm injection; LBR: live birth rate; VOC: volatile organic compound.
aWe conducted a PubMed search to identify relevant studies published in English until March 3rd, 2020 (start search date not specified). The search terms used were “air quality” AND “in vitro fertilisation” OR
“assisted reproductive technology.” We limited the search to full-text human studies that reported pregnancy outcomes, including cohort studies, case series, cross-sectional studies, and prospective studies.

R Sciorio, E Rapalini et al.
journals.sagepub.com/home/reh 9
other critical issues that might affect indoor air
quality and VOCs emissions, such as laboratory
furniture, wood, equipment, clothing and cosmet-
ics, and sterile plasticware, gloves, incubators and
numbers of embryologist working simultaneously
in the same area. Ideally, a risk management analy-
sis concerning laboratory air quality should take
into consideration not only to reduce but also to
avoid the risks associated with poor air quality con-
ditions.50 Fair evidence indicates that laboratory
air quality is associated with IVF outcome,
although the evidence is not unequivocal.46,47,51
Solid evidence based on prospective trials on the
optimal solution for AQ in ART is still missing.
Most published studies cited earlier are retrospec-
tive and lack proper controls. Some studies were
performed after laboratory renovation; therefore,
additional features might be changed, not only the
air filtration. However, evaluating the impact of
AQ on human early embryo development using
randomized controlled trials are not easy to per-
form due to technical and possible ethical implica-
tions. Furthermore, there are a multitude of factors
affecting ART results, including the characteristics
of treated patients, protocols and procedures, and
environmental pollutants. Finally, we do believe
that the optimal approach of AQ would involve an
embryology laboratory cleanroom, in which a mix-
ture of filtered fresh outside air and recirculated air
for PM and VOC air is supplied, and that includes
both the transfer and operating rooms. Such facil-
ity should be constructed and used in a way to
minimize the introduction, generation, and reten-
tion of particles and VOCs.
Author contributions
R.S., E.R., and S.C.E. contributed to the concep-
tion and designed the manuscript. R.S. and E.R.
wrote the first draft of the manuscript. R.S. and
S.C.E. wrote sections of the manuscript and
revised it for content. All authors contributed to
manuscript revision, read, and approved the sub-
mitted version.
Funding
The authors received no financial support for the
research, authorship, and/or publication of this
article.
Conflict of interest statement
The authors declared no potential conflicts of
interest with respect to the research, authorship,
and/or publication of this article.
ORCID iD
Romualdo Sciorio https://orcid.org/0000-0002
-7698-8823
Data availability statement
All data are included in the study.
References
1. Human Fertilisation Embryology Authority.
The UK reaches new milestones for fertility
treatments. https://www.hfea.gov.uk/about-us/
news-and-press-releases/2017-news-and-press-
releases/the-uk-reaches-new-milestones-for-
fertility-treatments/
2. De Geyter C, Calhaz-Jorge C, Kupka MS, etal.
ART in Europe, 2014: results generated from
European registries by ESHRE: the European IVF-
monitoring Consortium (EIM) for the European
Society of Human Reproduction and Embryology
(ESHRE). Hum Reprod 2018; 33: 1586–1601.
3. Mackenzie SC, Wickins-Drazilova D and Wickins
J. The ethics of fertility treatment for same-
sex male couples: considerations for a modern
fertility clinic. Eur J Obstet Gynecol Reprod Biol
2020; 244: 71–75.
4. Niederberger C, Pellicer A, Cohen J, etal. Forty
years of IVF. Fertil Steril 2018; 110: 185–324.e5.
5. Esteves SC and Bento FC. Implementation of
air quality control in reproductive laboratories
in full compliance with the Brazilian cells and
germinative tissue directive. Reprod Biomed Online
2013; 26: 9–21.
6. Conforti A, Mascia M, Cioffi G etal. Air
pollution and female fertility: a systematic review
of literature. Reprod Biol Endocrinol 2018; 16: 117.
7. Esteves SC and Bento FC. Clean room
technology and IVF outcomes: Brazil. In: Esteves
SC, Varghese AC and Worrilow KC (eds) Clean
room technology in ART clinics: a practical guide.
1st ed. Boca Raton, FL: CRC Press, 2017, pp.
371–392.
8. Worrilow KC, Huynh HT, Gwozdziewicz JB,
etal. A retrospective analysis: the examination of
a potential relationship between particulate (P)
and volatile organic compound (VOC) levels in
a class 100 IVF laboratory cleanroom (CR) and
specific parameters of embryogenesis and rates of
implantation (IR). Fertil Steril 2001; 76: S15–S16.
9. Brown SK. Chamber assessment of formaldehyde
and VOC emissions from wood-based panels.
Indoor Air 1999; 9: 209–215.

10 journals.sagepub.com/home/reh
Therapeutic Advances in Reproductive Health 15
10. Lundgren B, Jonsson B and Ek-Olausson B.
Materials emission of chemicals—PVC flooring
materials. Indoor Air 1999; 9: 202–208.
11. Srivastava PK, Pandit GG, Sharma S,
etal. Volatile organic compounds in indoor
environments in Mumbai, India. Sci Total Environ
2000; 255: 161–168.
12. Cohen J, Gilligan A, Esposito W, etal. Ambient
air and its potential effects on conception in vitro.
Hum Reprod 1997; 12: 1742–1749.
13. Gilligan A, Schimmel T, Esposito B Jr., etal.
Release of volatile organic compounds such as
styrene by sterile petri dishes and flasks used for
in-vitro fertilization. Fertil Steril 1997; 68(Suppl.
1): S52–S53.
14. Hall J, Gilligan A, Schimmel T, etal. The origin,
effects and control of air pollution in laboratories
used for human embryo culture. Hum Reprod
1998; 13(Suppl. 4): 146–155.
15. Rubes J, Selevan SG, Evenson DP, etal. Episodic
air pollution is associated with increased DNA
fragmentation in human sperm without other
changes in semen quality. Hum Reprod 2005; 20:
2776–2783.
16. Morbeck DE. Air quality in the assisted
reproduction laboratory: a mini-review. J Assist
Reprod Genet 2015; 32: 1019–1024.
17. Worrilow KC, Huynh HT, Bower JB, etal.
A retrospective analysis: seasonal decline in
implantation rates (IR) and its correlation with
increased levels of volatile organic compounds.
Fertil Steril 2002; 78: S39.
18. Forman M, Sparks A, Degelos S, etal.
Statistically significant improvements in clinical
outcomes using engineered molecular media
and genomically modeled ultraviolet light for
comprehensive control of ambient air (AA)
quality. Fertil Steril 2014; 102: e91.
19. Doshi A, Karunakaran S, Worrilow KC, etal.
What makes an IVF lab successful? In: Varghese
AC, Sjoblom P and Jayaprakasan K (eds) A
practical guide to setting up an IVF laboratory.
London: Jaypee Brothers Medical Publishers,
Ltd, 2013, pp. 13–23.
20. Mortimer DA, Cohen J, Mortimer ST,
etal. Cairo consensus on the IVF laboratory
environment and air quality: report of an
expert meeting. Reprod Biomed Online 2018; 36:
658–674.
21. Sandle T. Clean room design and principles.
In: Esteves SC, Varghese AC and Worrilow
KC (eds) Clean room technology in ART clinics:
a practical guide. 1st ed. Boca Raton, FL: CRC
Press, 2017, pp. 75–90.
22. Sadir RA, Velardes CJ and Sadir RI. What is
HEPA? How to achieve high efficiency particulate
air filtration. In: Esteves SC, Varghese AC and
Worrilow KC (eds) Clean room technology in ART
clinics: a practical guide. 1st ed. Boca Raton, FL:
CRC Press, 2017, pp. 55–63.
23. Fox JT. Volatile organic compounds: mechanisms
of filtration. In: Esteves SC, Varghese AC and
Worrilow KC (eds) Clean room technology in ART
clinics: a practical guide. 1st ed. Boca Raton, FL:
CRC Press, 2017, pp. 65–74.
24. Chiang YC, Chiang PC and Huang CP. Effects
of pore structure and temperature on VOC
adsorption on activated carbon. Carbon 2001; 39:
523–534.
25. Hodgson AI, Destaillats H, Sullivan DP, etal.
Performance of ultraviolet photocatalytic
oxidation for indoor air cleaning applications.
Indoor Air 2007; 17: 305–316.
26. Destaillats H, Sleiman M, Sullivan DP, etal.
Key parameters influencing the performance of
photocatalytic oxidation (PCO) air purification
under realistic indoor conditions. Appl Catal
B-Environ 2012; 128: 159–170.
27. Hay SO, Obee TN and Thibaud-Erkey C. The
deactivation of photocatalytic based air purifiers
by ambient siloxanes. Appl Catal B-Environ 2010;
99: 435–441.
28. Forman M, Polanski V, Horvath P, etal.
Reductions in volatile organic compounds,
aldehydes, and particulate air contaminants in an
IVF laboratory by centralized and standard alone
air filtration systems. Fertil Steril 2004; 82(Suppl.
2): S324.
29. Lawrence C. VOC levels in a new IVF laboratory
with both central and in-laboratory photocatalytic
air purification units. Alpha Sci Reprod Med 2007;
36: 1–5.
30. Bento FC and Esteves SC. Role of quality
manager in clean room assisted reproductive units
and policies for efficient running. In: Esteves SC,
Varghese AC and Worrilow KC (eds) Clean room
technology in ART clinics: a practical guide. 1st ed.
Boca Raton, FL: CRC Press, 2017, pp. 323–332.
31. Boone WR, Johnson JE, Locke AJ, etal.
Control of air quality in an assisted reproductive
technology laboratory. Fertil Steril 1999; 71:
150–154.
32. Heitmann RJ, Hill MJ, James AN, etal. Live
births achieved via IVF are increased by

R Sciorio, E Rapalini et al.
journals.sagepub.com/home/reh 11
improvements in air quality and laboratory
environment. Reprod Biomed Online 2015; 31:
364–371.
33. Mehta JG and Varghese A. Gases for embryo
culture and volatile organic compounds. In:
Esteves SC, Varghese AC and Worrilow KC
(eds) Clean room technology in ART clinics: a
practical guide. 1st ed. Boca Raton, FL: CRC
Press, 2017, pp. 99–117.
34. Sciorio R and Smith GD. Embryo culture at
a reduced oxygen concentration of 5%: a mini
review. Zygote 2019; 27: 355–361.
35. Wirka KA and Morbeck D. Sealed culture
systems with time-lapse video monitoring. In:
Esteves SC, Varghese AC and Worrilow KC
(eds) Clean room technology in ART clinics: a
practical guide. 1st ed. Boca Raton, FL: CRC
Press, 2017, pp. 353–360.
36. Santos AL. Clean room certification in assisted
reproductive technology clinics. In: Esteves SC,
Varghese AC and Worrilow KC (eds) Clean
room technology in ART clinics: a practical guide.
1st ed. Boca Raton, FL: CRC Press, 2017, pp.
217–236.
37. Brauer M and Griesinger F. Testing and
monitoring VOCs and microbials. In: Esteves
SC, Varghese AC and Worrilow KC (eds) Clean
room technology in ART clinics: a practical guide.
1st ed. Boca Raton, FL: CRC Press, 2017,
pp. 237–250.
38. Racowsky C, Hendricks RC and Baldwin
KV. Direct effects of nicotine on the meiotic
maturation of hamster oocytes. Reprod Toxicol
1989; 3: 13–21.
39. Fabro S. Penetration of chemicals into the
oocyte, uterine fluid, and preimplantation
blastocyst. Environ Health Perspect 1978; 24:
25–29.
40. Schimmel T, Gilligan A, Garrisi GJ, etal.
Removal of volatile organic compounds from
incubators used for gamete and embryo culture.
Fertil Steril 1997; 68(Suppl. 1): S165.
41. Nijs M, Franssen K, Cox A, etal. Reprotoxicity
of intrauterine insemination and in vitro
fertilization-embryo transfer disposables and
products: a 4-year survey. Fertil Steril 2009; 92:
527–535.
42. Otsuki J, Nagai Y and Chiba K. Peroxidation of
mineral oil used in droplet culture is detrimental
to fertilization and embryo development. Fertil
Steril 2007; 88: 741–743.
43. Hughes PM, Morbeck DE, Hudson SB,
etal. Peroxides in mineral oil used for in vitro
fertilization: defining limits of standard quality
control assays. J Assist Reprod Genet 2010; 27:
87–92.
44. Morbeck DE and Leonard PH. Culture systems:
mineral oil overlay. Methods Mol Biol 2012; 912:
325–331.
45. Munch EM, Sparks AE, Duran EH, etal.
Lack of carbon filtration impacts early embryo
development. J Assist Reprod Genet 2015; 32:
1009–1017.
46. Mortimer DA. Critical assessment of the impact
of the European Union Tissues and Cells
Directive (2004) on laboratory practices in
assisted conception. Reprod Biomed Online 2005;
11: 162–176.
47. Hartshorne GM. Challenges of the EU “tissues
and cells” directive. Reprod Biomed Online 2005;
11: 404–407.
48. European Union. Commission Directive
2006/86/EC implementing Directive
2004/23/EC of the European Parliament
and of the Council as regards traceability
requirements, notification of serious adverse
reactions and events and certain technical
requirements for the coding, processing,
preservation, storage and distribution of
human tissues and cells. Official Journal
of the European Union L294/32. https://
eur-lex.europa.eu/legal-content/EN/
TXT/?uri=CELEX%3A32006L0086
49. Esteves SC and Bento FC. Implementation
of cleanroom technology in reproductive
laboratories: the question is not why but how.
Reprod Biomed Online 2016; 32: 9–11.
50. Esteves SC and Bento FC. Air quality control
in the ART laboratory is a major determinant of
IVF success. Asian J Androl 2015; 18: 596–599.
51. Von Wyl S and Bersinger NA. Air quality in the
IVF laboratory: results and survey. J Assist Reprod
Genet 2004; 21: 283–284.
52. Khoudja RY, Xu Y, Li T, et al. Better IVF
outcomes following improvements in laboratory air
quality. J Assist Reprod Genet 2013; 30: 69–76.
53. Agarwal M, Chattopadhyay R, Ghosh S, et al.
Volatile organic compounds and good laboratory
practices in the in vitro fertilization laboratory:
the important parameters for successful outcome
in extended culture. J Assist Reprod Genet 2017;
34: 999–1006.
Visit SAGE journals online
journals.sagepub.com/
home/reh
SAGE journals