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Decisions for the IVF laboratory: Comparative analysis of embryo culture

incubators

Article in Reproductive Biomedicine Online · May 2014

DOI: 10.1016/j.rbmo.2014.01.004

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Reproductive BioMedicine Online (2014) 28, 535– 547

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SYMPOSIUM: QUALITY MANAGEMENT IN ASSISTED REPRODUCTIVE TECHNOLOGY

Decisions for the IVF laboratory: comparative

analysis of embryo culture incubators
Jason E Swain

Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI 48108, United States; Fertility Lab
Sciences, Englewood, CO 80112, United States
E-mail address: [email protected]

Jason E Swain, PhD, HCLD is currently the Corporate Laboratory Director of Fertility Lab Sciences in Lone Tree,
CO, USA. Formerly an associate professor in obstetrics and gynaecology and scientific director of Assisted
Reproduction Technology Laboratories at the University of Michigan, Jason completed his BSc at Hillsdale
College in his native Michigan, his MSc in animal science at Purdue University, his PhD in molecular and
integrative physiology at the University of Michigan and his clinical post-doctoral training in a private fertility
clinic in San Antonio, TX. Jason’s research interests include pursuit of methods to improve in-vitro embryo
culture conditions through modification of both the physical and chemical culture environment.

Abstract Incubators in the IVF laboratory play a pivotal role in providing a stable and appropriate culture environment required for
optimizing embryo development and clinical outcomes. With technological advances, several types of incubators are now available
and careful consideration is required for selection. Examination of variables, such as recovery/stabilization of temperature, gas
atmosphere and humidity, as well as understanding various approaches utilized by each device to regulate these variables, is crit-
ical. Additionally, a comprehensive examination of clinical studies that compare various incubators may provide insight into their
efficacy. Other factors, both technical and practical, must also be considered when selecting an incubator. Importantly, proper
management, including patient volume and workflow, is paramount in optimizing function of any incubator, regardless of the tech-
nology incorporated. This review highlights incubator function and reviews key environmental variables controlled and the technol-
ogy utilized in various units. Additionally, existing comparative studies focused on incubator recovery and clinical outcomes are
critically analysed. Finally, strategies employed for incubator management, as well as future potential incubator improvements
are discussed. While existing reports indicate that smaller benchtop/topload incubators provide faster recovery of environmental
variables, there is no clear advantage of any particular incubator based on clinical outcomes. RBMOnline
ª 2014, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

KEYWORDS: benchtop, blastocyst, box, embryo, incubator, topload

Introduction for embryo development and to achieve maximal assisted

reproductive outcomes. Key environmental variables to
Minimizing environmentally induced stress within the IVF consider within the culture system include pH of the culture
laboratory is crucial in creating a culture system optimized medium, temperature, media osmolality and air quality.

http://dx.doi.org/10.1016/j.rbmo.2014.01.004
1472-6483/ª 2014, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
 Author's personal copy

536 JE Swain

Importantly, all of these potential stressors can be Gas monitoring and recovery
impacted by the laboratory incubator. As a result, the incu-
bator is arguably the most important piece of equipment in A critically important function of the laboratory incubator is
the laboratory, controlling multiple environmental variables to reliably provide an appropriate gas atmosphere. Specifi-
and housing the embryos for the vast majority of their time cally, regulation of CO2 concentration is of paramount
in vitro. Thus, incubator selection and management is crit- importance, as this gas helps regulate pH of the culture
ical to ensure success of an IVF programme. medium. pH of the culture medium is an important variable
With advances in technology, multiple incubator types that can significantly impact gamete function and embryo
exist with varying capabilities and differing methods of reg- development (Swain, 2012). Additionally, reduced O2 con-
ulating their internal environment (Table 1). As a result, centration in the culture environment has, for many years,
selection of an appropriate culture incubator for the IVF repeatedly been found beneficial for both animal and
laboratory has become a complex process. The objective human embryo development and outcomes (Bavister, 2004;
of this review is to summarize key aspects of incubator func- Bontekoe et al., 2012; Mantikou et al., 2013), most notably
tion, critically examine comparative studies on incubator when used throughout the entire culture period to the blas-
performance in the laboratory, provide insight into proper tocyst stage (Kovacic and Vlaisavljevic, 2008; Meintjes
incubator management and discuss possible future incuba- et al., 2009; Waldenstrom et al., 2009). Thus, modern IVF
tor advancements. incubators should monitor and regulate both CO2 and O2
concentrations.
Incubator function Accurate and rapid monitoring of gas concentrations by
the incubator is critical to achieve target values in a timely
The primary function of an incubator within the IVF labora- fashion and ensure appropriate growth conditions. Central
tory is to provide a stable environment to optimize gamete to this function is the type of gas sensor used. The primary
function and embryo development in vitro. To achieve this method utilized by IVF incubators to monitor CO2 concen-
goal, an incubator must regulate several environmental tration includes one of two sensor types; thermal conductiv-
variables, including gas concentrations, temperature and ity (TC) or infrared (IR). Thermal conductivity sensors
humidity. Importantly, a variety of approaches and technol- function through measurement of resistance between two
ogies are utilized by different incubators to regulate these thermistors, with one enclosed and the other exposed to
environmental variables and each has benefits and disadvan- the incubator chamber (Chou, 1999a). The presence of
tages that should be considered when selecting a unit. Addi- CO2 in the incubator chamber changes the resistance
tionally, other functional and practical considerations exist between the two thermistors and permits elucidation of
that require consideration before implementation of the gas concentrations. Importantly, the resistance, and there-
equipment into the IVF laboratory. fore CO2 readings, of incubators using TC sensors are

Table 1 Incubator technology variables that should be considered when evaluating and selecting a unit for the laboratory.

Gas type CO2 sensor O2 sensor Temperature Designb Humidity Contamination Other
controla control controla,c

CO2-only Infrared Zirconium Air jacket Benchtop Yesd Heat Data logging
x xxxx
Low O2 – mixer Thermal Galvanic Water jacket Two-chamber No UV Cost
conductivity (fuel-cell)
x xxxx
Low O2 – x x Direct heat Multichamber x H2O2 Patient
premixed capacity
cylinder
x x x x Other (i.e. time- x Copper alloy Service
lapse imaging)
x xxxx
x x x x Small box x External HEPA Technology
integration
x x x Large box x x x
HEPA = high-efficiency particulate absorption.
a
May be influenced by presence/absence of an internal fan.
b
Other novel designs exist, but these are general terms to refer to the most commonly used incubators in the IVF laboratory; actual volumes
will vary from unit to unit.
c
Ease of removing inner parts and/or wiping interior is also important to consider.
d
Some units bubble gas through a water pan to expedite rehumidification.
 Author's personal copy

Embryo culture incubators 537

impacted by temperature and humidity. Conversely, IR sen- bonate and/or protein concentrations, which might
sors are largely humidity and temperature independent. require differing CO2 concentrations to achieve the desired
Infrared sensors emit a light and utilize optics to detect IR pH. Similarly, different laboratories may require different
absorbance, which is relative to the concentrations of CO2 CO2 concentrations, as variables such as laboratory eleva-
in the chamber atmosphere (Chou, 1999b). As a result, incu- tion can impact pCO2 and resulting pH. Once an appropriate
bators utilizing TC CO2 sensors tend to take longer to stabi- gas mixture is determined based on the requirements of a
lize gas atmosphere following door opening compared with particular culture medium and/or laboratory, accurate mix-
incubators outfitted with IR sensors, as CO2 concentrations ing may be verified through routine pH testing, independent
cannot be fully determined and subsequently adjusted until gas measurement or through a formal certificate of analyses
both temperature and humidity stabilize. Thus, considering supplied by the vendor.
improvements to IR sensor lifespan and reduction in cost, Independent of gas sensor or gas supply type, incubator
incubators outfitted with IR sensors have become the pre- volume also influences gas equilibration and recovery tim-
ferred option in many laboratories to help to improve envi- ing. Following door opening, traditional ‘large-box’ incuba-
ronmental stability and recovery. tors (150–200 l) may require an extended time to refill
Similarly to incubator CO2 sensors, two primary types of
sensor are used to assess incubator O2 concentration; gal-
vanic/fuel cell or zirconium sensors (Hitech Instruments Table 2 Approximate culture chamber volumes of various
Technical Note). A galvanic sensor is a diffusion-limited incubators/devices utilized for clinical IVF.
metal/air battery (Chou, 1999a). Oxygen diffuses through
Model Approximate chamber
the outer barrier of the sensor to reach the inner cathode
volume (l)
where it is reduced to hydroxyl ions which, in turn, oxidize
the metal anode. A current, proportional to the rate of con-
sumption of O2, is generated when the cathode–anode cir- K-systems G185 0.299 · 10 chambers
cuit is completed. The rate of O2 diffusion to reach the Astec EZ Culture 0.310 · six chambers
cathode and the cell current is a direct function of this dif- Cook K-MINC 0.43 · two chambers
fusion rate, this in turn being a direct function of the con- Planar BT-37/INC-A20 0.43 · two chambers
centration of O2 in the sample. A zirconium sensor is an Labotect Labo C-Top 0.5 · two chambers
impervious tube with a zirconia element with a closed end Astec IVF Cube 0.775 · four chambers
and is coated externally and internally with porous metal ESCO Miri Multiroom 0.886 · six chambers
electrodes. At elevated temperatures, the element Fertilitech Embryoscope 2.4
becomes an O2-ion conductor, which results in a voltage Billups-Rotheburg MIC-101 5.8
being generated between the electrodes. The value of the Modular Chamber
voltage is dependent upon the differences between the Galaxy 14S 14
partial pressures of the O2 in the sample and the O2 in a ref- Labotect C-16 16
erence gas (generally air). Although more recent galvanic Astec Penguin DH 30
sensors have improved the rapidness of their response read- Astec CCM IVF 30
ing, they tend to yield slower response times compared with Astec APC30/APM30 32
zirconium sensors and more frequent replacement is often IKS IVS-9000 33
required to ensure adequate functioning. IKS INB-203 42
Importantly, for both incubator O2 and CO2 readings, Galaxy 48R 48
external incubator display values should not be relied upon Panasonic MCO 5M 48
to determine atmospheric recovery during re-equilibration, Binder CB 53 53
as some incubator displays return to their programmed set Astec APC50/APM50 57
points prior to actual re-equilibration of internal gas con- Labotect C-60 60
centrations. A more accurate assessment or comparison of Memmert Inc108med 108
gas recovery rate entails independent measurement of O2 Binder CB 150 150
and CO2 concentrations using real-time measuring devices Forma series II 3130 150
placed inside the incubator chamber. Heracell 150i 150
In addition to the two primary sensor types used to mea- Memmert Inc153med 153
sure CO2 and O2 concentrations, accurate gas concentra- Astec CDI/SMA 163
tions can be achieved in the absence of gas sensors Galaxy 170R 170
through the use of supply cylinders of premixed gas. Cylin- Panasonic MCO 18/19 170
ders of premixed gas can be supplied directly to an incuba- IKS INB-203XL 179
tor or to a sealed modular chamber placed inside the Nuaire Autoflow NU4950 188
incubator, rather than requiring the incubator to mix the Ruskinn Ac-tive IVF 394
gases into the proper ratios. Using this approach, appropriate A complete list of all incubators is not feasible. This list focuses on
CO2/O2 concentrations are quickly achieved as soon as the currently produced incubators with low-O2 capability commonly
incubator volume has been filled with the premixed gas. sold for use in IVF laboratories. Volumes were obtained from
However, proper quality control is required to ensure that manufacturer/sales websites, product literature or via communi-
the premixed gas concentrations/ratios within the supply cation with company technical personnel. In addition to chamber
cylinder yield the desired medium pH and growth condi- volume and number, ability to minimize incubator opening/closing
tions. Different culture media may contain different bicar- is important for atmospheric stability.
 Author's personal copy

538 JE Swain

with CO2 and/or nitrogen gases. These large units were ini- be problematic. An emerging approach to improve air qual-
tially designed for use with multiple flasks of somatic cells ity to some incubators includes recirculating atmosphere
placed onto each shelf. However, dishware used for IVF is past a UV light source for photocatalytic breakdown of VOCs
considerably smaller, few dishes are used, and often only (Chapuis et al., 2002; Sharmin and Ray, 2012). Whether one
one patient is placed onto a single shelf at any given time. of the aforementioned approaches to remove VOCs is supe-
Thus, ‘small-box’ incubators (14–48 l) have come into rior to another or provides superior results in the IVF labora-
use and, depending on workflow, may help improve gas tory incubator is unknown.
recovery, reduce environmental stress and improve embryo It should be mentioned that incubators that utilize cylin-
development compared with larger incubators (Avery and ders of premixed gas have the ability to filter the entirety of
Greve, 1992). More recently, benchtop/topload units of the gas supply prior to it entering the incubator chamber.
varying sizes/configurations designed specifically for clini- Incubators that mix gases themselves, either CO2-only or
cal IVF have extremely small chambers (0.31–0.5 l), fur- low-O2 incubators, have at least some portion of room air
ther improving gas atmosphere recovery time (Table 2). present, although if room air is of high quality this likely
Of note, while smaller incubator volumes are beneficial in poses little problem. Also important to note, plasticware
terms of rate of atmospheric recovery following repeated or internal incubator components may de-gas inside the
opening, large volume isolator-based incubator systems elevated temperatures of the incubator chamber (Cohen
can also yield acceptably high outcomes, likely due, in part, et al., 1997). Thus, despite having acceptable outside air
through limiting or eliminating excursions from the enclosed quality or a prefiltered gas supply, VOCs may still be present
system (Hyslop et al., 2012). Thus, as will be discussed, inside any incubator. In these cases, proper initial cleaning
incubator management is also a key component for opti- of incubators and de-gassing of devices and supplies may
mized incubator function. help address concerns. Additionally, placement of modular
VOC filter units in the incubator chamber or recirculation
Air quality of chamber atmosphere through external filters may also
be effective.
An additional variable related to gas atmosphere that
impacts incubator function is air quality. Air quality, specif- Temperature
ically presence and amounts of volatile organic compounds
(VOC), may compromise embryo development (Cohen Another primary function of the laboratory IVF incubator is
et al., 1997; Hall et al., 1998; Khoudja et al., 2013; Merton to maintain an appropriate temperature for gamete func-
et al., 2007), although the relevant concentrations of VOC tion and embryo development. It is also well known that
are still unknown. As a result, most laboratories have dedi- temperature can impact various aspects of gamete and
cated air handling systems to filter out particulates as well embryo function, most notably meiotic spindle stability (Sun
as VOCs and various studies indicate a benefit to embryo et al., 2004; Wang et al., 2001, 2002) and possibly embryo
development and/or outcomes once air quality is improved metabolism (Leese et al., 2008). However, data indicate
(Boone et al., 1999; Khoudja et al., 2013). However, while that temperature gradients may exist in the female repro-
outside air quality may be important, it is the quality of ductive tract (Hunter, 2012; Hunter and Einer-Jensen, 2005;
the atmosphere inside the incubator that is likely of more Hunter et al., 2006). Thus, while the optimal target temper-
concern. ature for IVF incubators that contain varying cell types and
Outside air quality has an obvious impact on atmospheric embryos at different developmental stages is still unknown
quality within the incubator, especially in CO2-only incuba- (Higdon et al., 2008; Hong et al., 2012) and beyond the
tors, which carry a balance of 94% room air. However, the scope of this review, maintaining an accurate temperature
quality of gas from the supply tanks must also be consid- while inside the incubator is mandatory for reducing envi-
ered, especially in low-O2 incubators, which flood their inte- ronmental stress.
riors with nitrogen from these tanks to reduce O2 Three main types of heating approaches are employed by
concentration to 5%. VOCs have been detected in gas sup- IVF incubators. Two common warming methods used pri-
ply tanks used for IVF incubators (Hall et al., 1998). In these marily in box-type incubators include a water jacket or air
cases, filtering the supply gases through inline filters prior to jacket, both of which warm the air in the incubator chamber
incubator entry may be an effective approach to improve and may or may not include an internal fan to circulate. The
incubator atmosphere. These inline filters not only contain third heating approach used by benchtop/topload units
high-efficiency particulate absorption (HEPA) filtration to entails contact of the warmed incubator surface, upper
reduce particle counts but also methods to reduce VOC as and lower, and direct heat transfer to the culture dish and
well, including activated charcoal or potassium permanga- enclosed medium. Importantly, each incubator warming
nate. At least one preliminary study has shown improve- approach has benefits and limitations. Water-jacketed incu-
ments in embryo development and outcomes following bators retain heat longer in case of incubator opening or
implementation of inline gas-filtration systems (Esteves power failure. However, units are heavy, tend to have
et al., 2006). Placement of specialized VOC filtration units higher power consumption, which may burden emergency
inside incubators can also improve air quality and outcomes power supplies, and are accompanied by concerns that con-
(Mayer et al., 1999; Merton et al., 2007; Schimmel et al., tamination may originate from inside the water jacket.
1997), although this is not always the case (Battaglia Air-jacketed incubators warm up quickly, but do not retain
et al., 2001; Higdon et al., 2008; McLellan et al., 2001) and, heat for long periods. Air-jacketed, but not water-jacketed,
depending on their size, fitting into smaller incubators may units are also compatible with heat-sterilization decontam-
 Author's personal copy

Embryo culture incubators 539

ination cycles and may help with contamination concerns. reduce chances of contamination. Various incubators are
Finally, direct heat/contact results in very rapid heat recov- constructed with copper-containing alloys, as copper can
ery following opening of the unit or dish removal, but main- act as antimicrobial and antifungal agent (Borkow and Gab-
tenance of this temperature for any period of time can be bay, 2004; Grass et al., 2011). However, at least one study
problematic if power interruption occurs. Of note, while suggested that oxidized copper particles from incubator
not necessarily a component of the incubator per se, any walls may have detrimental effects on bovine embryo devel-
incubator can and should be connected to commercially opment (Avery and Greve, 1992), although experimental
available battery backup units and/or generator-protected design precluded any conclusive correlation and several
electrical outlets to avoid concerns associated with power copper-containing incubators are used successfully for
loss. human embryo culture. Alternatively, some air-jacketed
Temperature gradients can exist in any incubator, incubators feature heat decontamination cycling capability.
regardless of the warming system used. Such occurrences Other incubators can be outfitted with hydrogen peroxide
are most commonly noted in box-type incubators utilizing sterilization capability by the manufacturer. Ultraviolet
air- or water-jacketed warming. A preliminary report light treatment of water pans is also available to reduce
indicated slight temperature variations when culture incidence of contamination, although this feature is often
dishes were placed in various locations within a large-box turned off to avoid damage to cells within the incubator.
water-jacketed incubator, with measurements of 36.97C, Additionally, any incubator can be sterilized and/or cleaned
37.17C and 37.23C (Stoddart et al., 2003). Similar by removing inner pieces for autoclaving and wiping down
findings were also recently reported between two the interior of the unit with embryo-safe products, such as
large-box incubator, where minor (0.07–0.17C), but hydrogen peroxide or other commercial IVF cleaning solu-
significant temperature differences were identified tions, preferably with low VOC content. Units with fewer
between shelves and between incubators (Walker et al., removable shelves or lacking internal fans are easier to
2013). Whether such minor fluctuations have an impact clean and may help reduce the risk of contamination.
is unknown, but independent temperature measurement Quality control may be another variable to consider when
between shelves on box-type units, between individual selecting a laboratory incubator. Several optional features
culture chambers or across warmed surfaces of various may be available on units than can assist with routine mon-
benchtop/topload unit configurations can provide insight itoring, including data logging capabilities to monitor
into temperature accuracy, as well as possible variation real-time temperature fluctuations or number of door open-
that could impact gamete and embryo development and ings. Some incubator designs may make other aspects of
function. As will be discussed, examination of possible quality control more difficult, although perceived limita-
temperature gradients is useful in optimizing incubator tions can often be overcome. For example, in bench-
management. top/topload incubators, it may be difficult to measure pH
of the culture medium. However, specialized pH meters or
blood gas analysers can be purchased in some cases, or pH
Humidity can be tested directly from the gas supply if using a cylinder
of premixed gas. Alternatively, small test tubes can often
Many incubators regulate humidity to avoid media evapo- be laid inside units to permit medium equilibration for
ration during culture to avoid harmful rises in medium subsequent measurement.
osmolality that can compromise embryo development Finally, additional consideration must be given to cost
(Lane et al., 2008; Swain et al., 2012). This humidification and capacity of each incubator, as well as space occupied.
is usually supplied in a passive fashion, via evaporation of Several incubators are required in any IVF laboratory to help
a water reservoir commonly placed in the bottom of incu- avoid overcrowding and promote proper incubator
bator chambers. However, the presence of a water pan management, as well as provide backup capabilities in case
for humidity control is also a potential source of contam- of unit malfunction or scheduled downtime for routine
ination and should be monitored and replaced regularly. A maintenance.
variation on this approach includes bubbling inlet gases
through the water pan or a supplied water bottle, which Comparative studies and clinical outcomes
may help humidify the air more rapidly, but also acts as
an additional filter of inlet gases. Of note, humidity inside
the incubator is not necessarily required to culture While literature from various commercial companies can be
embryos if adequate amounts of oil overlay are used found demonstrating performance characteristics of a par-
and may be dependent upon the number of days of con- ticular incubator against a competing incubator or technol-
tinuous culture, although this should be validated within ogy, comparative studies within a laboratory and resulting
each laboratory. Furthermore, lack of humidity may ben- clinical outcomes reported in the peer-reviewed literature
efit incubators using TC CO2 sensors, as humidity recovery may offer better insight into incubator performance within
is no longer a limiting factor. the context of the IVF laboratory. Unfortunately, very few
studies comparing environmental stability and recovery of
particular incubator units exist, and even fewer studies
Other considerations comparing outcomes of embryo development or assisted
reproductive outcomes are available (Table 3). Further-
Other considerations for incubator selection include more, careful examination of the existing literature is
approaches available for cleaning and sterilization to required to understand why differences may exist, and scru-
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540 JE Swain

Table 3 Incubator comparison studies.

Study Incubator No. of No. of Method Outcome: results, Conclusions and

comparison patients embryos statistical significance notes

Cooke et al. MINC versus NA NA Compared time to MINC recovered Direct heat transfer
(2002) Forma large- temperature recovery temperature within of the benchtop unit
box (water from 35C to 37C of 5.5–6.5 min resulted in faster
jacketed, TC 1.0 ml media with depending on media temperature
CO2 sensor) 0.1 ml oil overlay volume recovery than the
versus 50 ll media indirect-warming
with 1 ml oil overlay system of the larger
incubator
x xxxx
x x x x x Forma did not recover An aluminium block
temperature by can help with
20 min for either temperature
volume (reached 36.2 recovery in the large
and 36.7C) indirect-warming
incubator
x xxxx
Fujiwara et al. K-MINC 30 334 RCT: sibling embryos Temperature recovery Benchtop incubators
(2007) benchtop versus randomized after 2PN time: 5 min versus offer improved
ASTEC small- check 30 min, P < 0.01 culture environment
box (TC CO2 recovery times
sensor, galvanic
O2 sensor,
water jacketed)
x x x x 5-s door opening, O2 recovery time: Recovery time may
repeated 10 times 3 min versus 8 min, influence the
and averaged P < 0.01 formation of good-
quality embryos
x x x x x Early ‘good’ embryo x
rate: 40% versus 38%,
P < 0.05
x x x x x ‘Good’ blastocyst x
formation rate: 15%
versus 8%, P < 0.05
x xxxx
Lee et al. K-MINC 97 1,189 RCT: sibling embryos Temperature recovery Benchtop incubators
(2010) benchtop versus randomized time: 1 min versus offer improved
Forma large- 180 min, P < 0.01 culture environment
box (high O2, TC recovery times
CO2 sensor,
water jacketed,
inner doors)
x x x x 10-s door opening and CO2 recovery time: No differences were
outcomes compared 8 min versus 120 min, found between
P < 0.01 incubators in respect
to embryo quality or
outcomes
x x x x x Humidity recovery: x
12 min versus 180 min,
P < 0.01
x x x x x Fertilization rate: 72% x
versus 67%, P < 0.05
x x x x x Day-3 grade, NS x
x x x x x Implantation rate: 39% x
versus 32%, NS
x x x x x Clinical pregnancy x
rate: 64% versus 54%,
NS
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Embryo culture incubators 541

Table 3 Incubator comparison studies (continued).

Study Incubator No. of No. of Method Outcome: Conclusions and notes

comparison patients embryos results,
statistical
significance

xxx
Cruz et al. Embryoscope 60 478 RCT: sibling Blastocyst No difference between the
(2011) versus Heracell embryos were formation Embryoscope and a standard
150 large-box randomized after rate: 55% incubator in blastocyst
(air jacketed, 2PN check versus 51%, formation blastocyst viability
high O2) NS rate or ongoing pregnancy
rate
x x x x Morphological Blastocyst Study also compared
assessment was viability: 29% Embryoslide individual
made at days 2 versus 35%, culture versus microdrop
and 3 NS individual culture
x x x x Embryos were not Ongoing x
removed from pregnancy
Embryoscope for rate: 43%
assessment (6/14) versus
42% (8/19),
NS
x xxxx
Kirkegaard Embryoscope 59 676 RCT: sibling No. of 4-cell No difference was found
et al. (2012) versus Galaxy R embryos were embryos on between the Embryoscope
170 large-box randomized after d2: NS and standard incubator for
(direct heat, insemination embryo development,
high O2, IR CO2 implantation rate or clinical
sensor) pregnancy rate
x xxxx
x x x x Morphological No. of 7–8- Study also compared
assessment at cell embryos Embryoslide individual
days 2, 3 and 5 on d3, NS culture in small microvolume
versus group culture in Nunc
wells of larger volume
x x x x Embryos were No. of x
removed at equal blastocysts
time points for on d5, NS
both incubators
and outcomes
compared
x x x x x Implantation x
rate: 37%
(7/19) versus
33% (6/18),
NS
x x x x xClinical pregnancy rate: 38% x
(8/21) versus 30% (7/23), NS

2PN = 2 pronuclei; IR, infrared; NA = not applicable; NS = not significant; RCT = randomized controlled trial; TC = thermal conductivity.

tiny often points out limitations in study design that should measured O2 recovery rather than CO2 recovery. While O2
be considered when interpreting results. and CO2 will recover at the same rate in the benchtop unit
For example, when comparing a small benchtop incuba- due to using a premixed gas supply, O2 will recover much
tor unit with two topload chambers (0.43 l) and a more slowly than CO2 in the frontload unit, which uses sep-
small-box incubator (32 l), it was found that, after a 5-s arate gas supplies due to the larger amount of nitrogen
opening, the benchtop/topload unit had improved temperature needed in the larger volume. It is unknown if such large
recovery (5 min versus 30 min), O2 recovery (3 min versus differences would exist when measuring CO2, which is likely
8 min), improved ‘good’ early embryo development (40% to be more important. Furthermore, in this case, the
versus 38%) and improved ‘good’ blastocyst formation (15% small-box unit was outfitted with outdated technology, uti-
versus 8%; Fujiwara et al., 2007). Interestingly, authors lized a TC CO2 sensor and was water jacketed. Whether the
 Author's personal copy

542 JE Swain

same differences would be apparent if using the faster IR Additionally, embryos were cultured individually in micro-
CO2 sensor and air-jacketed unit is unknown. Finally, no drops in the large-box incubator, while being placed into
oil overlay was used and overall blastocyst conversion rates individual microwells for the benchtop (M Cruz, personnel
in both incubators were low. It is possible that use of oil communication). This is important to note because the type
overlay would help stabilize pH and temperature and of culture dish/conditions can influence embryo develop-
perhaps improve the suboptimal growth conditions in the ment through modifications of the microenvironment (Swain
study. Thus, while the benchtop/topload unit likely recov- and Smith, 2011). While no significant difference between
ers atmosphere and temperature more rapidly, closer exam- the number of day-3 or day-5 transfers based on a particular
ination of the study reveals that the discrepancies between incubator was reported (benchtop/time-lapse versus stan-
the two incubators may not be as pronounced if compared dard incubator; Cruz et al., 2011), upon reanalysis of the
with an updated box-type incubator optimized technology. reported data using a different statistical program, it
A subsequent study again compared a box-type incubator appears that more day-5 transfers were performed from
and a small 2-chambered benchtop/topload unit, examining the larger boxincubator (34/58) compared with the smaller
CO2, temperature and humidity recovery as well as fertiliza- time-lapse incubator (19/50; chi-squared 0.052,
tion rate, embryo quality, clinical pregnancy and implanta- chi-squared with Yates correction 0.038, Fishers test
tion rates (Lee et al., 2010). Following a 10-s incubator P = 0.038, Fisher’s exact two-tailed P = 0.036). Often day-5
opening, it was found that there was a significant difference transfer is dictated by superior quality or number of
in temperature recovery (1 min versus 180 min), CO2 recov- embryos; although no differences in outcomes were noted.
ery (8 min versus 120 min) and humidity recovery (12 min Thus, use of smaller benchtop incubators do not necessarily
versus 180 min), with faster recovery occurring in the equate to better embryo quality, as several other variables
benchtop/topload unit. Interestingly, the large-box incuba- are involved in efficacy of the culture environment.
tor was outfitted with nonairtight inner doors which may not A similar study comparing a benchtop/time-lapse incuba-
provide as stable a gas environment as new units which tor versus a standard large-box unit (large-box incubator
employ air-tight inner doors. Again, the large-box incubator size confirmed via JJ Hindkjaer, personal communication)
used the slower TC sensor and was water jacketed. Finally, used embryos from infertile patients and compared embryo
the benchtop/topload unit utilized low-O2 culture via pre- development, pregnancy and implantation rates. Despite
mixed gas, while the large-box incubator utilized CO2 only. confounding variables, such as use of different culture
As mentioned previously, low O2 appears to yield better dishes (Embryoslide versus Nunc 4-well) and embryo density
embryo development and outcomes (Bavister, 2004; Bonte- (single versus group), no significant differences in any end-
koe et al., 2012; Mantikou et al., 2013). Furthermore, the point were found between incubators (Kirkegaard et al.,
use of the cylinder of premixed gas in the benchtop/topload 2012). While high O2 was used in each incubator (K Kirkeg-
unit may provide improved air quality over use of 94% aard, personal communication), other conditions in each
room air in the large-box incubator. Indeed, one preliminary specific incubator, such as pH or humidity, were not
study comparing the same types of large-box and small reported. Failure to properly control all variables between
benchtop/topload incubators reported that air quality/gas incubators, which is a difficult task, makes it hard to deter-
composition may be partially responsible for improved mine ‘superiority’ of a particular incubator. Thus, while
mouse blastocyst development observed in two out of five these studies help demonstrate safety of time-lapse imaging
different culture media in the benchtop unit compared with of embryos and use of a novel benchtop/time-lapse incuba-
the large-box incubator, although why benefit was not tor, it could also be used to demonstrate that a large-box
observed in the other three media is unclear and other vari- incubator, with proper management, can yield similar out-
ables that could impact outcomes between incubators also comes to a benchtop unit using time-lapse imaging. Indeed,
existed (Morbeck et al., 2011). These same confounding if limiting patient number and the associated door openings
variables exist in another preliminary study that compared of large-box incubators, with proper management, tradi-
the same large-box and small benchtop unit (Mortimer tional box-type incubators can perform similarly compared
et al., 2003) and make it impossible to truly determine with benchtop/time-lapse units (M Tucker and M VerMilyea,
the impact of the incubator alone. Importantly, despite personnel communication). However, in this case, several
the differences in the culture parameters and suboptimal large-box incubators are needed to facilitate the required
culture conditions provided in the large-box incubators in management and match the capacity of the benchtop unit.
these studies, there were no reports of significant differ- A more recent retrospective observational multicentre
ence in human embryo development, clinical pregnancy or cohort study comparing clinical pregnancy rates from
implantation rates (Lee et al., 2010). embryos grown in a benchtop/time-lapse incubator com-
In a comparative examination of incubators using oocytes pared with a large-box CO2 incubator with a TC sensor, dem-
from donors, outcomes between a benchtop/time-lapse onstrated a 20.1% increase in clinical pregnancy rate per
imaging incubator and a standard large-box incubator were oocyte retrieval or 15.7% per transfer (Meseguer et al.,
assessed (large-box incubator size confirmed via M Cruz, 2012). However, as pointed out by the authors in their dis-
personnel communication). Despite significant differences cussion, this could be due to a variety of factors including,
in embryo handling, including not removing embryos from but not limited to, improved embryo selection from use of
the benchtop incubator while removing embryos at least time-lapse imaging and not removing embryos for daily
twice from the large-box incubator, as well as use of low observation from the benchtop/time-lapse unit. Interest-
O2 in the benchtop unit but not the large-box incubator, ingly, in this study, air quality in the smaller incubator,
authors reported no significant difference in blastocyst for- but not the large-box incubator, was extensively filtered
mation, quality or ongoing pregnancy (Cruz et al., 2011). through HEPA and activated carbon and was UV-treated
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Embryo culture incubators 543

every 10 min. An improved approach to assess the impact of properly control other confounding variables, such as
the incubators themselves may include comparison of out- patient number per incubator, making it very difficult to
comes using time-lapse imaging inside a large-box incuba- determine the true impact of a particular incubator.
tor, with similar filtering and air quality to those from a
benchtop incubator with time-lapse imaging.
Incubator management
A comparison of a small two-chamber benchtop/topload
unit (0.43 l) using direct heat versus a large-box (170 l)
incubator using a water jacket and no inner doors demon- Following critical review of existing comparative studies, it
strated a significantly faster recovery of temperature in becomes apparent that it is impossible to determine the
the benchtop/topload direct heat unit (Cooke et al., 2002). ‘best’ incubator. This will vary from laboratory to labora-
Temperature in the benchtop/topload unit recovered to tory based on specific use and needs. Certainly results can
37C within 5.5–6.5 min, dependent upon the volume of vary between incubators for a variety of reasons (Avery
medium tested, while the large-box incubator failed to and Greve, 1992; Higdon et al., 2008). This reinforces the
reach the set point following 20-min recovery (36.2C and need for strict quality control as well as proper management
36.7C). Whether the same would hold true with an air-jack- of laboratory IVF incubators to optimize their functions and
eted box-type incubator, small or large sized, or with units maximize outcomes (Higdon et al., 2008). Insight into spe-
using sealed inner doors is unknown. Interestingly, use of cific incubator units, both benchtop/topload and standard
milled aluminium blocks to hold culture dishes within the box type, their functioning and potential drawbacks can
box-type incubator were able to maintain temperature be found elsewhere (Meintjes, 2012). Regardless of the spe-
and help lessen/avoid temperature decrease in the culture cific model of incubator utilized within the laboratory, with-
dish (Cooke et al., 2002). This demonstrates the importance out proper incubator management, environmental stability
of incubator management in optimizing performance of var- and embryo development can be compromised in even the
ious units. most cutting-edge unit employing the newest technology.
Finally, a more recent preliminary study retrospectively Proper incubator management includes various approaches
analysed laboratory data following equipment change and aimed at maintaining environmental stability inside the
suggests that mini bench-top incubators can significantly unit. This includes distribution of patient samples and
improve laboratory outcomes compared with single- proper workflow to avoid overuse of any particular incuba-
chambered large-box incubators (Hill et al., 2013). When tor. Overuse of an incubator results in the inability to main-
changing from 12 large-box incubators using low O2 to 16 tain a stable culture environment due to repeated
bench-top incubators using cylinders of premixed gas opening/closing. Thus, management requires an adequate
(CO2/O2/N2 6:5:89), a significant improvement in 2 number of incubators based not only on total cycle volume,
pronuclei formation was observed, with no significant but also on the time frame of when these cycles are per-
improvement in blastocysts frozen, cycles with embryos fro- formed. For example, an IVF laboratory that performs 200
zen or donor pregnancy rate. However, again, differences in cycles spread over the entire year will have a different incu-
air quality with differing gas supplies could be a possible bator requirement than an IVF laboratory that performs the
explanation for results. Perhaps more likely, with the addi- same 200 cycles in four batches a year because the workload
tion of four additional benchtop units compared with the for a single incubator will be greater for the laboratory that
large-box-type incubators, undoubtedly fewer patients batches. The number of incubators required can be deter-
were placed into each bench-top incubator. Thus, it is not mined through analysing a particular laboratory’s workflow,
possible to determine if one incubator type/technology taking into account how many patients can fit into an indi-
was actually superior to the other, as differences in patient vidual unit (i.e. one patient per shelf or two patients per
distribution between incubators impacts opening/closing unit), how many patients will be seen within a period of
and environmental stability. time (i.e. 6 d) and other relevant variables.
Thus, in examining existing comparative studies on incu- In addition to an appropriate number of incubators,
bators, differences likely exist between environmental workflow between incubators must also be considered. Pre-
recovery between various units, including gas atmosphere ferred use of a single incubator over others due to a conve-
and temperature. This depends largely on the size of the nient location can compromise the environmental stability
incubator, but also on the technology incorporated into of the individual incubator due to increased opening/
the unit, such as gas sensor type or temperature regulation closing. It was demonstrated that reducing door opening
approach. However, careful attention should be paid to the from six to four times over a 6-d period on a small-box incu-
use of optimal available technology/approaches for each bator utilizing a water jacket with TC CO2 and galvanic O2
type of incubator to better analyse the comparisons being sensors resulted in significantly improved human blastocyst
made. Many existing studies compare newer smaller bench- formation (53% versus 43%) and good-quality blastocyst
top units to older outdated large-box units. While this rates (60% versus 51%), although no differences in day-3
reflects many real-world system changes, comparison of embryo quality, implantation or clinical pregnancy rates
new smaller units to an ‘optimized’ large-box or small-box were observed (Zhang et al., 2010). Also supporting the ben-
unit might be more insightful. Additionally, it becomes efit of reduced incubator opening and improved embryo
apparent that, while smaller units recover gas atmosphere development, use of a gassed/sealed modular chamber
and temperature more rapidly, and this undoubtedly results placed inside the incubator to stabilize gas atmosphere
in less environmental stress imposed upon the system, this resulted in significantly improved mouse blastocyst develop-
may not necessarily equate to better outcomes. Further- ment and increased cell number compared with embryos
more, the available comparative incubator studies fail to cultured in a standard box incubator opened approximately
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544 JE Swain

11 times per day (Gardner and Lane, 1996). Similar improve- vibrating motor, similar to those used to vibrate cellular
ments in mouse embryo development and clinical outcomes phones, attached to the area housing the embryo dish to
were also observed with use of a large enclosed isola- provide gentle mechanical stimulation for brief periods
tor-based culture system, likely due, in part, to improved between image capture. Prior studies indicate that 5 s of
environmental stability due to reduced incubator opening vibration at 44 Hz improved embryo development and out-
(Hyslop et al., 2012). Finally, limiting patient number in comes (Hur et al., 2013; Isachenko et al., 2010, 2011).
small two-chambered benchtop/topload units to a maxi- Accordingly, incubators could be modified to incorporate
mum of four patients, compared with the full unit capacity these devices, utilizing the strengths of both large-box
of eight patients, has been used to improve blastocyst for- and benchtop units, including housing each in a small, inde-
mation (M Tucker, M Vermilyea, W Caswell, personal com- pendently sealed chamber. Indeed, at present there are
munication). Thus, IVF cases should be distributed already incubators that utilize individual or removable
between all available incubators to avoid overuse or exces- chambers, which remain sealed/gassed and separate from
sive opening, regardless of the size or format of the the rest of the larger unit, thereby permitting isolation of
incubator. individual patient material while not compromising the gas
Other approaches to avoid excessive incubator opening environment of other patient samples when accessed. Some
include the use of ‘holding’ incubators that can be used of these even include real-time imaging. Furthermore, reli-
for transient procedures, such as dish equilibration, sperm ance on tanks of medical gases for incubators could poten-
swim-up/capacitation and even brief culture of thawed tially be alleviated through use of a safe, clean, chemically
embryos prior to same-day/immediate transfer. Using older supplied atmosphere. As demonstrated by prior studies sup-
‘outdated’ incubators for these purposes may help avoid plying CO2 from a simple citric acid and sodium bicarbonate
overuse of incubators that are used primarily for extended reaction in a closed test-tube system, proper pH can be
embryo culture. obtained and maintained over extended periods of time,
Finally, use of various commercially available adjuncts permitting production of mouse blastocysts at similar rates
can help with incubator management and maximize envi- and with similar cell numbers to embryos grown in standard
ronmental stability. Various gas or air filters have been men- large-box incubators (Swain, 2010, 2011). This approach
tioned and can be used to improve air quality. Use of inner would be conducive to simplifying the incubator system
doors on box-type incubators can help prevent excessive gas and offers a means of promoting low-cost IVF (Swain, 2011).
exchange. Desiccator jars or modular chambers can main- Indeed, this method was later adopted for clinical use as
tain gas atmosphere within box-type incubators during part of a low-cost IVF initiative (Klerkx et al., 2013; Van
repeated opening/closing. Additionally, use of specialized Blerkom et al., 2013). Authors demonstrated that fertiliza-
milled aluminium blocks designed to hold culture dishes tion and embryo development could proceed in test tubes
can help maintain a stable temperature during door opening filled with a chemically generated atmosphere and the
or during routine observations performed outside the approach has resulted in seven live births. Similar
incubator. approaches may result in more cost-effective modular cul-
ture devices, perhaps further lending itself to individualized
Incorporating new technology culture platforms to help stabilize the growth environment.

Another important consideration for incubator selection Conclusions

entails the ability to implement new technology. Recent
advances in dynamic embryo culture include motorized tilt- Incubator selection is an important decision for the IVF lab-
ing devices, vibrating platforms and even piezo-actuated oratory, as these devices regulate several environmental
pin systems (Smith et al., 2012; Swain and Smith, 2011; variables that can impact embryo development. While novel
Swain et al., 2013), all which require standard box-type culture approaches may reduce the need for traditional
(large or small) incubators for placement. Similarly, some incubators (Blockeel et al., 2009; Hyslop et al., 2012; Itoi
emerging real-time embryo imaging devices that use et al., 2012; Ranoux and Seibel, 1990; Ranoux et al., 1988;
portable modular cameras also require large- or small-box Suzuki et al., 1999; Swain, 2010, 2011; Taymor et al., 1992;
incubators for implementation (Chavez et al., 2012; Cona- Vajta et al., 1997, 2004; Van Blerkom et al., 2013; Varisanga
ghan et al., 2013; Wong et al., 2010). With proper manage- et al., 2000), for the time being they remain a central part
ment, box-type incubators used to accommodate these of a modern IVF laboratory. Functional aspects of the incu-
devices aimed at improving embryo development and/or bator, such as gas capability and sensor type, as well as
selection can likely perform in a similar fashion as benchtop temperature control and size/patient capacity need to be
units. Similarly, with continued development, perhaps novel considered. Smaller incubator units, especially bench-
dynamic culture devices can be scaled down to permit top/topload devices, result in faster gas atmosphere and
incorporation into small benchtop/topload incubators. temperature recovery. However, no study has clearly dem-
Looking towards the future, incubator size and design onstrated a distinct advantage of any specific incubator
will likely continue to change. An emerging trend for bench- type in terms of human embryo development or clinical out-
top/topload units is apparent and further modifications or comes. Regardless of the unit, low-O2 capability should be
advancements may continue to help revolutionize and drive available and utilized and a IR CO2 probe is preferable for
incubator design. For example, emerging time-lapse imag- those units that mix the gases to permit the fastest CO2
ing devices could potentially be modified to incorporate recovery. Practical issues, such as cost and space, must also
dynamic vibrational culture. One could envision a small be weighed. The proper number and type of incubators to
 Author's personal copy

Embryo culture incubators 545

adequately support a laboratory’s caseload must be Cohen, J., Gilligan, A., Esposito, W., Schimmel, T., Dale, B., 1997.
determined on a laboratory-by-laboratory basis. A mix of Ambient air and its potential effects on conception in vitro.
incubator types, including both large-box, small-box and Hum. Reprod. 12, 1742–1749.
benchtop/topload within a laboratory helps cover multiple Conaghan, J., Chen, A.A., Willman, S.P., Ivani, K., Chenette, P.E.,
Boostanfar, R., Baker, V.L., Adamson, G.D., Abusief, M.E.,
scenarios and offers several options for utilization, includ-
Gvakharia, M., Loewke, K.E., Shen, S., 2013. Improving embryo
ing implementation of emerging technologies. selection using a computer-automated time-lapse image analysis
Paramount in appropriate functioning and optimizing test plus day 3 morphology: results from a prospective multi-
incubator performance is proper incubator management. center trial. Fertil. Steril. 100, 412–419.
Regardless of incubator size or the technology incorpo- Cooke, S., Tyler, J.P., Driscoll, G., 2002. Objective assessments of
rated/utilized, improper management of case workflow or temperature maintenance using in vitro culture techniques. J.
failure to perform proper quality control can compromise Assist. Reprod. Genet. 19, 368–375.
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caseload to help avoid overcrowding and maintain a stable blastocyst and ongoing pregnancy rates in oocyte donation
patients whose embryos were monitored by time-lapse imaging.
environment. As technology continues to advance and new
J. Assist. Reprod. Genet. 28, 569–573.
culture platforms and embryo selection technologies Esteves, S.C., Verza Jr., S., Gomes, A.P., 2006. Comparison
become available, incubators will undoubtedly need to be between international standard organization (ISO) type 5 and
adapted to meet the changing needs of the field. type 6 cleanrooms combined with volatile organic
compounds filtration system for micromanipulation and embryo
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