The Effects of Chemical and Physical Factors On Mammalian Embryo Culture and Their Importance For The Practice of Assisted Human Reproduction

Human Reproduction Update, Vol.22, No.1 pp.
2 –22, 2016
Advanced Access publication on July 22, 2015 doi:10.1093/humupd/dmv034
The effects of chemical and physical
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factors on mammalian embryo culture
and their importance for the practice of
assisted human reproduction
Petra L. Wale 1,2,* and David K. Gardner 1
1
School of BioSciences, University of Melbourne, Parkville, Victoria, Australia 2Melbourne IVF, Melbourne, Victoria, Australia
*Correspondence address. E-mail: [email protected]
Submitted on February 10, 2015; resubmitted on May 28, 2015; accepted on June 25, 2015
table of contents
...........................................................................................................................
† Introduction
† Methods
† Chemical factors
Oxygen
Ammonium
Volatile organics
Albumin; blood versus recombinant
† Physical factors
Temperature and pH
Oil overlay
Incubation volume/embryo density
Pipetting induced shear stress
Static nature of culture
Light
† Conclusions
background: Although laboratory procedures, along with culture media formulations, have improved over the past two decades, the issue
remains that human IVF is performed in vitro (literally ‘in glass’).
methods: Using PubMed, electronic searches were performed using keywords from a list of chemical and physical factors with no limits placed
on time. Examples of keywords include oxygen, ammonium, volatile organics, temperature, pH, oil overlays and incubation volume/embryo
density. Available clinical and scientific evidence surrounding physical and chemical factors have been assessed and presented here.
results and conclusions: Development of the embryo outside the body means that it is constantly exposed to stresses that it would
not experience in vivo. Sources of stress on the human embryo include identified factors such as pH and temperature shifts, exposure to atmos-
pheric (20%) oxygen and the build-up of toxins in the media due to the static nature of culture. However, there are other sources of stress not
typically considered, such as the act of pipetting itself, or the release of organic compounds from the very tissue culture ware upon which the
embryo develops. Further, when more than one stress is present in the laboratory, there is evidence that negative synergies can result, culminating
in significant trauma to the developing embryo. It is evident that embryos are sensitive to both chemical and physical signals within their micro-
environment, and that these factors play a significant role in influencing development and events post transfer. From the viewpoint of assisted
human reproduction, a major concern with chemical and physical factors lies in their adverse effects on the viability of embryos, and their
& The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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Effects of chemical and physical factors on embryos 3
long-term effects on the fetus, even as a result of a relatively brief exposure. This review presents data on the adverse effects of chemical and
physical factors on mammalian embryos and the importance of identifying, and thereby minimizing, them in the practice of human IVF. Hence,
optimizing the in vitro environment involves far more than improving culture media formulations.
Key words: ammonium / embryo culture / combined stress / density / oxygen / pH / pipetting / temperature
perceived as sources of stress on gametes and embryos, they will not
Introduction be covered here.
Assisted reproductive techniques (ART) have advanced significantly over
the last three decades resulting in higher implantation and take home
baby rates. Further improvements in preimplantation embryo culture Methods
systems should continue to augment success rates, while reducing the
This review presents the available evidence regarding the effect of the listed
number of cycles required in achieving a pregnancy. To date such
chemical and physical factors on mammalian embryo culture and their im-
improvements in culture conditions have come largely in the form of portance for the practice of assisted human reproduction. A search was per-
enhanced media formulations, with relatively little attention being paid formed in PubMed regarding these chemical and physical factors with no
to the fact that one is trying to maintain the early events of life outside limits placed on time. Keywords used included oxygen, ammonium, volatile
of the human body (Gardner, 2008). Fertilization and embryo develop- organics, temperature, pH, oil overlays and incubation volume/embryo
ment in vitro have the potential to introduce (often inadvertently) stresses density. Available clinical and scientific evidence surrounding these physical
which cannot only impair embryo development in the laboratory, but and chemical factors have been assessed, interpreted and presented here.
also which can have down-stream effects after transfer (Lane and
Gardner, 1994). In vivo, the developing preimplantation embryo is
exposed to gradients of nutrients, hormones, cytokines and growth Chemical factors
factors as it progresses through the fallopian tube to the uterus
(Gardner et al., 1996; Hannan et al., 2011; Thouas et al., 2015). Within Oxygen
the lumen of the female tract the embryo resides in a few hundred nano- Analysis of oxygen within the oviduct and uterus of different mammalian
litres of a complex viscous fluid (Hoversland and Weitlauf, 1981; Salleh species has shown that the concentration of oxygen is typically 2– 8% in
et al., 2005), characterized by high levels of mucins, albumin and glycosa- luminal fluids (Bishop, 1957; Mastroianni and Jones, 1965; Ross and
minoglycans (Lee and Ax, 1984; Leese, 1988; Laurent et al., 1995; Zorn Graves, 1974; Maas et al., 1976; Fischer and Bavister, 1993). These
et al., 1995), and by reduced levels of oxygen (typically 2–8%, Fischer and levels are in contrast to that present in air (20% depending upon alti-
Bavister, 1993). The embryo is in constant motion, moved by gentle cili- tude). The first report of the beneficial effects of physiological oxygen
ated and muscular action of the female tract, and metabolites produced (5%) for preimplantation embryo culture was published over 40
by the embryos are removed from its immediate vicinity due to its prox- years ago by Wes Whitten. Whitten, using both inbred and hybrid
imity to the epithelia of the female tract and hence maternal circulation. mice strains, observed that whilst all 1-cell mouse embryos developed
This scenario is in stark contrast to the laboratory environment, where in an oxygen concentration of 5%, all arrested when the oxygen concen-
typically gametes and embryos are exposed to relatively large volumes tration was 20% (Whitten, 1969). Whitten went on to state that ‘mouse
of culture medium (up to 100 ml per embryo, Bolton et al., 2014), embryos do have a low, but definite, oxygen requirement and that both
remain static during culture, while resting on a polystyrene substrate, oxygen lack and excess may damage the surface membrane’. Con-
and create unstirred layers where the end products of metabolism consequently, the gas mix of 5% O2, 5% CO2 and 90% N2 was adopted as
centrate and nutrients become rate limiting (Gardner and Lane, 2012, a standard in embryo culture (Whitten, 1971). Concomitantly,
2014). The culture dishes themselves can release volatile organic com- Edwards et al. (1970) reported the development of the human
pounds, which can adversely affect embryo developmental potential embryo under different oxygen conditions (5 and 20%). Comparisons
(Holyoak et al., 1996). Manipulation of gametes and embryos in the la- were made across different media, within each oxygen concentration
boratory is facilitated through pipetting, an act which if performed too fertilization rates of 10.3% for 5% oxygen and 11.9% for 20% oxygen,
vigorously can induce stress-activated responses (Xie et al., 2007). and cleavage rates of 90.9% for the 5% oxygen group and 87.5% for
During embryo manipulations both temperature and pH of the the 20% oxygen group were observed (Edwards et al., 1970). In a follow-
medium can drift, which can negatively impact gametes and embryos. up study demonstrating the growth of human blastocysts in culture, it
In addition, if more than one stress is present in the laboratory, then was stated: ‘we seem to have achieved this improved embryonic develop-
there is evidence that negative synergies can result, culminating in signifi- ment through better handling of the cultures than previously . . . under
cant trauma to the developing embryo (Awonuga et al., 2013). reduced oxygen tension’ (Steptoe et al., 1971). Hence, based on the devel-
From the viewpoint of human ART, the major interest in chemical and opment seen in these early two studies, an environment of 5% oxygen was
physical factors lies in their adverse effects on the viability of embryos, adopted as the standard gas mixture for subsequent cultures (Steptoe and
even if exposure is for a relatively brief period of time. This review, there- Edwards, 1978; Edwards et al., 1981; Edwards and Steptoe, 1983).
fore, presents data on the adverse effects of chemical and physical factors Given that relative oxygen concentration was shown to be an import-
on mammalian embryos and their importance in the practice of human ant factor for the development of cultured mouse embryos in these pio-
IVF and embryo culture. Although assisted fertilization through ICSI, neering studies (Whitten, 1969, 1971), a number of investigators
embryo biopsy for genetic screening and cryopreservation can all be followed up and examined the effects of oxygen on other mammalian
4 Wale and Gardner
species. Tervit et al. (1972) endeavoured to overcome the in vitro block in These data reveal that oxygen can influence mouse embryo develop-
development of sheep embryos at the 8- to 16-cell stage by culturing ment at both the pre- and post-compaction stages (Wale and
embryos in a range of oxygen concentrations. Embryos cultured in 5% Gardner, 2010), and that the detrimental effects of 20% oxygen on the
oxygen developed through the reported 8- to 16-cell block in develop- early embryo were not only irreversible but cumulative (Fig. 1a and b).
ment, with a higher proportion of late morulae and blastocysts (56%) Kirkegaard et al. (2013) subsequently replicated these study conditions
compared with results from 10 and 20% oxygen concentrations (16 with human embryos, with the early exposure to 20% oxygen reduced
and 0%, respectively) (Tervit et al., 1972). Building upon this, Quinn to 24 h. Similar results were seen, with delayed development after
and Harlow (1978) examined development of inbred mouse embryos culture in 20% oxygen, with the pre-compaction embryo unable to
under an atmosphere containing 0, 2.5, 5, 10 or 40% oxygen and in agree- recover from exposure to 20% oxygen, and culture in 20% O2 reducing
ment with Whitten’s findings, they determined that 5% oxygen sup- developmental rates and delaying the completion of the third cell cycle
ported optimal embryo development. Of interest, the culture of (Kirkegaard et al., 2013).
mouse embryos in 20% oxygen resulted in blastocysts with fewer blasto- Such data raise the question; how does atmospheric oxygen impair
meres than those that had developed in 5% oxygen (Quinn and Harlow, embryo development? Extensive analyses have revealed that oxygen reg-
1978; Harlow and Quinn, 1979). More recently Karagenc et al. (2004) ulates events during the preimplantation period, and affects all aspects of
demonstrated that although oxygen concentration had no effect on cell function. Analysis of mouse blastocyst gene expression (Gardner and
inbred mouse embryo implantation or resultant fetal weights, embryos Lane, 2005; Rinaudo et al., 2006) demonstrated that atmospheric
cultured in the presence of 20% oxygen resulted in a dramatic decrease oxygen has a causal role. When Gardner and Lane (2005) cultured
in fetal development per blastocyst and fetal development per implant- zygotes from inbred mice to the blastocyst stage in complex medium
ation, compared with embryos cultured in 5% oxygen.
Over the intervening three decades several studies have gone on to
demonstrate that culture in a reduced oxygen concentration, of 5– 7%,
improves preimplantation embryo development in a multitude of mam-
malian species including the sheep (Thompson et al., 1990), cow
(Thompson et al., 1990), goat (Batt et al., 1991) pig (Berthelot and
Terqui, 1996) and outbred mice (Gardner and Lane, 1996). Further-
more, there is a growing body of evidence that the preimplantation
embryo exhibits stage-specific sensitivity (a phenomenon highlighted
throughout this review) to stress, including oxygen. Embryos exposed
to atmospheric oxygen as pronucleate oocytes for as little as one hour
followed by culture in 5% oxygen displayed delayed development at
the morula stage (Pabon et al., 1989) and blastocyst stage (Umaoka
et al., 1992). Subsequently, Gardner and Lane (1996) determined that
even the equilibration of culture media for 6 h in atmospheric oxygen
prior to culture in 5% oxygen was detrimental to subsequent embryo de-
velopment (reflecting the extended time it takes to equilibrate media
with 5% oxygen, which is considerably longer than that required for
carbon dioxide equilibration). Consistent with these data, Karagenc
et al. (2004) reported that exposure of pronucleate oocytes to atmos-
pheric oxygen for 23 h resulted in a significant decrease in blastocyst
cell numbers.
In order to further investigate the temporal responses of the preim-
plantation embryo to oxygen concentrations in vitro, Wale and
Gardner (2010) used time-lapse microscopy to continuously assess
embryo development. Mouse preimplantation embryos were cultured Figure 1 (a) Effect of oxygen on mouse blastocyst development
in 5 or 20% oxygen concentrations for the first 48 h, followed by rates. Embryos were cultured in either 5 or 20% oxygen for 48 h and
culture in the same or reciprocal oxygen concentrations for another then either transferred to the same or different oxygen tension, creating
48 h, resulting in four treatments: group 1 (control, 5 and 5%); group 2 four groups of embryos. Significantly different from embryos incubated
(5 and 20%); group 3 (20 and 5%); and group 4 (20 and 20%). Compared in control (5 and 5%): **P , 0.01, ***P , 0.001. Significantly different
with embryos cultured in 5% oxygen, embryos cultured in 20% oxygen from embryos incubated in 5 and 20% treatment: @P , 0.05,
@@
were delayed at the first cleavage by 0.45 h, at the second cleavage by P , 0.01. Data from Wale and Gardner (2010). (b) Effect of
0.84 h and at the third cleavage by 3.19 h. Of great interest, switching oxygen on mouse blastocyst total cell numbers. Embryos were cultured
from 20 to 5% oxygen after 48 h could not alleviate the perturbations in either 5 or 20% oxygen for 48 h and then either transferred to the
same or different oxygen tension, creating four groups of embryos. Sig-
induced during the cleavage stages, indicating that either the pre-
nificantly different from embryos incubated in control treatment:
compaction stage is more sensitive to oxygen stress, or that the
*P , 0.05, ***P , 0.001. Significantly different from embryos incubated
damage imparted by 20% oxygen to the cleavage stage embryos is irre- in 5 and 20% treatment: @@P , 0.01. Data from Wale and Gardner
versible. Partial or complete culture in 20% oxygen resulted in a signifi- (2010).
cant reduction in blastocyst cell numbers compared with control.
under 20% oxygen, 19 genes were up-regulated and 12 were down- SA-b-gal and g-H2A.X compared with blastocysts cultured in 20%
regulated compared with the 5% oxygen group. Rinaudo et al. (2006) cul- oxygen. Meuter et al. (2014) concluded that energy dependent cell
tured outbred mouse zygotes to the blastocyst stage in simple and responses to stress, particularly to DNA damage, may lead to cell
complex medium, under both 5 and 20% oxygen. Embryos cultured in cycle arrest and ultimately may affect viability or long-term development.
5% oxygen displayed fewer perturbations in their global pattern of This is relevant to human ART as these data suggest that the senescence-
gene expression, and embryos cultured in complex medium showed like phenotype is largely due to culture in atmospheric oxygen (Meuter
gene expression patterns more closely resembling those of the in vivo et al., 2014).
controls. Li et al. (2014a) subsequently examined the effects of atmos- Consistent with transcriptomic and proteomic analyses, atmospheric
pheric oxygen on global methylation patterns in bovine embryos. Com- oxygen also induces metabolic perturbations during the preimplantation
pared with embryos cultured at 5% oxygen, embryos cultured at 20% period. The first evidence came from Khurana and Wales (1989) who
exhibited a significant increase in global DNA methylation at the 4-cell demonstrated that culture of mouse embryos in 5% oxygen was asso-
and blastocyst stages, indicating that oxidative stress can induce ciated with higher levels of glucose oxidation. Wale and Gardner
changes in the embryonic epigenome. Additionally, chronic exposure (2012) investigated the effects of oxygen on carbohydrate and amino
to 20% oxygen induces irreversible X chromosome inactivation (XCI) acid utilization, both of which are linked to the ability of the human and
in human embryonic stem cells (hESC). In contrast, hESC isolated mouse embryo to develop in culture (Gardner et al., 2001; Houghton
under 5% oxygen do not undergo premature XCI and are more able et al., 2002) and to subsequent viability of embryos after transfer (Lane
to retain pluripotency (Lenger et al., 2010). Of note, XCI is considered and Gardner, 1996; Brison et al., 2004; Gardner et al., 2011). The pre-
a model epigenetic event (Whitelaw, 2006; Lee, 2011). More recently and post-compaction periods of embryo development were again of
it has been determined that culture in an atmosphere of 5% oxygen is interest, as the cleavage stage embryo displayed a greater sensitivity to
required for epigenetic stability in hESC, and that culture in the presence oxygen, and distinct differences were recorded in embryonic metabolism
of 20% oxygen leads to aberrant methylation of the DLK1-DIO3 (Delta- before and after compaction. Consistent with the biphasic results during
like homologue 1/type 3 iodothyronine deiodinase) gene cluster, an embryo culture (Wale and Gardner, 2010) the metabolic response of the
imprinted region (Xie et al., 2014). Defects in this imprinted region are embryo to the oxygen concentration employed was different between
associated with developmental delay, mental retardation and even post- pre- and post-compaction stages (Wale and Gardner, 2012). When cul-
natal death in humans (Mo et al., 2015). tured in 20% oxygen the cleavage stage embryo exhibited higher rates of
Of note, changes in the transcriptome do not always directly reflect pyruvate uptake and amino acid turnover compared with embryos cul-
changes in cellular function, nor do they account for post-translational tured in 5% oxygen. Interestingly, the increase in amino acid turnover
modifications or protein–protein interactions. Consequently Katz-Jaffe by cleavage stage embryos as a result of culture in 20% oxygen could
et al. (2005) investigated the effects of oxygen on the embryonic prote- be largely accredited to higher amino acid consumption.
ome. Consistent with transcriptomic data, protein profiles from In contrast, post-compaction embryos cultured in 20% oxygen exhib-
embryos cultured at 5% oxygen concentration more closely resembled ited lower rates of glucose uptake and a lower uptake of most amino acids
profiles from in vivo developed embryos, whereas those blastocysts (Wale and Gardner, 2012). The rate and fate of glucose consumed was
developed in atmospheric oxygen exhibited a divergent proteome. also different: blastocysts cultured in 20% oxygen displayed higher rates
Looking at specific proteins, Rodina et al. (2009) determined that of lactate production from glucose, i.e. a high rate of glycolysis. Of note,
culture of bovine embryos in 5% oxygen, but not 20%, maintained blasto- low rates of glycolysis are consistent with blastocyst viability (Gardner
cyst viability and facilitated constitutive and inducible interferon-tau pro- and Wale, 2013; Lee et al., 2015). That 5% oxygen is associated with
duction, which promotes uterine implantation. It is important to lower rates of glycolysis in the blastocyst is consistent with the data of
consider that although both transcriptomic and proteomic data indicate Khurana and Wales (1989), i.e. 5% oxygen is linked with higher levels
that embryos that developed in 5% oxygen are similar to those devel- of glucose oxidation. The carbohydrate and amino acid data from
oped in vivo, the in vivo-derived blastocysts have to be flushed from the Wale and Gardner (2012) support the hypothesis that viability
uterus. Consequently they will experience some stress during the collec- depends not only on the amount (or rate) of nutrient(s) consumed
tion through to their extraction/analysis. How much this alters the true (Renard et al., 1980; Gardner and Leese, 1987; Brison et al., 2004),
in vivo phenotype is unknown and impossible to determine with current but also on which metabolic pathway (or fate) the embryo uses to me-
technologies. tabolize nutrients (Lane and Gardner, 1996; Gardner and Wale, 2013;
More recently Meuter et al. (2014) demonstrated that the oxygen con- Lee et al., 2015). Furthermore, these findings again highlight differences
centration employed during preimplantation embryo culture induces in the physiology of the embryo before and after compaction, and the
markers of permanent cell cycle arrest (cellular senescence). Mouse embryo’s increased susceptibility to stress during the cleavage stages.
blastocysts were derived both in vitro under 5 or 20% oxygen, and Given that metabolism is a biomarker of viability (Gardner et al., 2011;
in vivo. Blastocysts were assessed for primary markers of senescence: Gardner and Wale, 2013), it is of significance that atmospheric oxygen
the phosphorylated histone family member H2A.X (g-H2A.X), a has such a profound effect on metabolic function. Consistent with its
marker of DNA oxidative damage and senescence-associated effects on blastomere function, data indicate that embryo culture in a
b-galactosidase (SA-b-gal), as well as senescence-associated genes reduced oxygen environment has benefits post-partum (Iwata et al.,
p21, p16, and interleukin 6. Compared with in vivo-derived blastocysts, 2000; Fischer-Brown et al., 2005).
in vitro embryos (from both oxygen concentrations) had higher levels Significantly, changes in cell function as a result of exposure to atmos-
of SA-b-gal, nuclear g-H2A.X, and p21 mRNA expression, indicating pheric oxygen are not limited to embryonic cells. Oxygen also regulates
that a senescence-like phenotype is induced by in vitro culture. the energy metabolism of hESC, and is intrinsic to the self-renewal of
However, blastocysts cultured in 5% oxygen had low levels of both hESC. The concentration of oxygen significantly affects hESC physiology
6 Wale and Gardner
with coordinated changes in gene expression, in the absence of detect- with time-lapse microscopy, to determine if the effects of oxygen on
able alterations in undifferentiated marker expression (Forristal et al., embryos were influenced by their sex. It was determined that atmos-
2013). Similarly, Harvey et al. (2014) reported that under atmospheric pheric oxygen induced significant delays in female embryo development
oxygen conditions glucose consumption was reduced in hESC. as early as the 7-cell stage, and that this persisted through blastocyst for-
In spite of the available data, clinical debate continues around whether mation (Gardner and Kelley, 2013). Recent data on the developmental
exposure to atmospheric oxygen throughout the preimplantation kinetics of human embryos cultured in atmospheric oxygen also show
period has a cumulative effect on the preimplantation embryo, or that male embryos develop faster than females in non-physiological con-
whether specific embryonic stages are more vulnerable to non- ditions (Bronet et al., 2015).
physiological oxygen. Nanassy et al. (2010) concluded that extended In light of the overwhelming experimental data that mammalian preim-
culture of the preimplantation embryo increases stress on the embryo plantation embryo development, physiology and viability are significantly
and, as such, reduced oxygen (5%) should be employed for culture of compromised by atmospheric oxygen, why was the practice of using a gas
the post-compaction embryo but not before. However, several phase of carbon dioxide in air adopted so widely for human IVF, and why
studies have reported no difference in blastocyst development rates has the employment of atmospheric oxygen persisted for so long? Sadly,
when the preimplantation embryo was only exposed to atmospheric there is not a clear-cut answer to this question, and perhaps one must
oxygen concentration at post-compaction stages (Karagenc et al., consider the technologies available in the early years of human IVF in
2004; Kind et al., 2005; Feil et al., 2006). When development of order to deduce how atmospheric oxygen became adopted as standard
human embryos to the blastocyst stage under reduced or atmospheric practice for so long. In the pioneering studies and early days of human IVF
oxygen was compared, both pregnancy and implantation rate increased and embryo culture, both atmospheric oxygen (Veeck et al., 1983;
as a result of the reduced oxygen environment (Catt and Henman, Wortham et al., 1983a,b) and 5% oxygen (De Kretzer et al., 1973;
2000). Whilst it is clear that embryos are able to form blastocysts Trounson et al., 1980; Lopata et al., 1982; Lopata, 1983) were employed.
when cultured in atmospheric oxygen, these data highlight the import- To create the stable atmosphere of 5% O2, 5% CO2 and 90% N2, pre-
ance of measuring the developmental competency and viability of the re- mixed gas-cylinders and desiccators, or equivalent sealable chambers,
sultant embryos to further evaluate the complete pathological response were used. Such chambers were cumbersome and consumed a lot of
of the embryo to culture conditions. This is especially true as the clinical space in each incubator. The rapid growth and widespread application
practice of selecting the highest quality embryos for transfer may contrib- of ART led to a practical move from desiccators/sealed chambers to
ute to the inconclusiveness of the human studies, as a comparison of incubators which could handle larger numbers of culture dishes for an
fresh pregnancy rates may not reflect the overall viability of a cohort. increasing number of patients’ embryos. In the late 1980s this was
In support of this, two prospective RCTs have demonstrated that the achieved using tissue culture incubators, designed to create an atmos-
culture of human embryos in low oxygen is associated with an overall in- phere of 5% CO2 in air. Given the relative paucity of data on the
crease in live births; Meintjes et al. (2009a) reported an increase from adverse effects of atmospheric oxygen on embryonic and fetal develop-
30.7 to 42.9% for implantation rate and 42.6 to 57.4% for live birth ment in the early years of human IVF, clinical IVF laboratories all over the
rate in 20 and 5% oxygen, respectively. Similarly, Waldenström et al. world adopted the use of atmospheric oxygen (20%). Fortuitously, the
(2009) reported a 10% increase in live birth rate when embryos were cul- last 10 –15 years have seen the development of excellent incubators
tured in 5% oxygen. A recent Cochrane evaluation of whether culturing capable of maintaining both carbon dioxide and oxygen concentrations.
preimplantation human embryos in a physiological oxygen compared to Such chambers include tissue culture-styled chambers, as well as new
atmospheric improves the treatment outcome concluded; ‘The results bench-top designs, including several that are also able to perform time-
of this systematic review and meta-analysis suggest that culturing lapse microscopy.
embryos under low oxygen concentrations improves the success rates Finally, given the documented negative impact that oxygen has on
of IVF/ICSI, resulting in an increase in the live birth rate’ (Bontekoe cell physiology, it would appear prudent to consider the inclusion of anti-
et al., 2012). This conclusion is consistent with the data from other oxidants in IVF/culture media, to ensure levels of protection are con-
species (mouse, cow, sheep, goat and pig) where employment of physio- ferred during the first days of life. Serendipitously, pyruvate, which is
logical oxygen concentration throughout development results in super- present in all culture media, is a powerful antioxidant itself (Andrae
ior embryo transfer outcomes. et al., 1985; Kouridakis and Gardner, 1995; O’Fallon and Wright,
Of additional interest are the observations that oxygen appears to 1995). Several antioxidants have now been shown to have protective/
have a greater detrimental effect on female embryos than male. It is stimulatory effects during embryo culture or cryopreservation including
well established that male and female embryos differ with respect to ascorbate (Umaoka et al., 1992; Lane et al., 2002), glutathione (Legge
gene expression, their proteome and metabolism (Review by Gardner and Sellens, 1991; Hansen and Harris, 2014), carnitine (Phongnimitr
et al., 2010, 2011). Consequently, it is not surprising that gender differ- et al., 2013; Takahashi et al., 2013), cysteine (Li et al., 2014b), cysteamine
ences exist in response to cellular trauma induced by 20% oxygen. (Elamaran et al., 2012) and lipoate (Linck et al., 2007). Their roles in
Meintjes et al. (2009b) observed a significant skewing in the sex ratio of human ART wait to be fully elucidated.
babies following embryo culture in atmospheric oxygen and blastocyst Given that there are no longer any technical restrictions in using
transfer, with significantly more males (58.5%) being born. However, physiological levels of oxygen (i.e. below 10%), together with the sub-
when human embryos were cultured in the presence of 5% oxygen, stantial data from animal models covering all aspects of embryo physi-
the sex ratio at birth was restored to 51.9% males (Meintjes et al., ology, it is time to confine the use of atmospheric oxygen to the annals
2009b). Subsequently, Gardner and Kelley (2013) used a mouse of human IVF. Of note, the vast majority of studies on lower oxygen
model, with males carrying an X chromosome with a green fluorescent levels for embryo culture have focused on 5%. However, the optimum
protein tag to facilitate sex determination by fluorescence, combined oxygen concentration for human embryo development has yet to be
elucidated, and further, we do not know whether stage-specific differ- effects of ammonium. Glutamine is the greatest contributor to the am-
ences exist. monium pool in vitro, as the most labile amino acid, and in early formula-
tions of human embryo culture media was typically present at 1–2 mM,
thereby representing a source of significant levels of ammonium in vitro.
Ammonium The substitution of this amino acid with the more stable dipeptide forms
Amino acids are abundant in the fluids of the female reproductive tract alanyl-glutamine or glycyl-l-glutamine has greatly reduced the accumula-
(Fahning et al., 1967; Perkins and Goode, 1967; Menezo and Laviolette, tion of ammonium in modern embryo culture media (Gardner and Lane,
1972; Casslén, 1987; Miller and Schultz, 1987; Gardner and Leese, 1990; 2003; Biggers et al., 2004). Interestingly, it is also possible to transaminate
Moses et al., 1997; Harris et al., 2005), and hence are key components of enzymatically the ammonium in situ to the non-toxic glutamate (Lane and
mammalian embryo culture media (Gardner, 2008). Amino acids fill key Gardner, 1995). Ammonium build-up can be further reduced by renewal
physiological niches as development and differentiation proceed, includ- of the culture medium after 48 h of culture (Gardner and Lane, 1993;
ing acting as osmolytes (Dumoulin et al., 1992; Lawitts and Biggers, 1992; Lane and Gardner, 1995). In reality, production of ammonium during
reviewed by Baltz, 2001), as buffers of internal pH (Edwards et al., 1998a) embryo culture cannot be prevented. However, considerable efforts
and the regulation of carbohydrate metabolism (Gardner and Sakkas, have been made to ensure that the concentration of ammonium stays
1993; Lane and Gardner, 2005). Prior to the 2-cell stage, the inability below 75 mM (the lowest concentration reported to have an adverse
of the pre-compaction embryo to utilize lactate as the sole energy effect on the cleavage stage human embryo) by titrating down the
source can be attributed to low activity of the malate-aspartate levels of amino acids present in culture medium and substituting glutam-
shuttle. It has been determined that aspartate is the rate-limiting factor ine with heat stable di-peptide forms. It is not known at what concentra-
affecting the activity of the malate-aspartate shuttle and that the addition tion ammonium has no detectable impact on blastomere function, nor
of exogenous aspartate (10 mM) enables the mouse zygote to utilize has a systematic study been performed to evaluate the importance of
lactate in the absence of pyruvate and continue development (Lane the frequency of medium renewal, i.e. 24 versus 48 h. Interestingly,
and Gardner, 2005). Inclusion of amino acids in modern culture media this study may not be forthcoming with the move to time-lapse micros-
has, therefore, in no small way contributed to the steady increase in copy and uninterrupted culture. At least the move to use a reduced
success rates for human IVF over the past two decades (Bavister, oxygen concentration during embryo culture can help alleviate the
1995; Gardner and Lane, 1997; Devreker et al., 2001; Gardner, 2008). impact of ammonium (as outlined below).
Unfortunately, there is a downside to the inclusion of amino acids due Similar to the stress associated with atmospheric oxygen, it is the pre-
to their lability. compaction embryo which appears most sensitive to ammonium. Even a
When present in any culture medium and incubated at 378C, amino short exposure during the cleavage stages can result in significant disrup-
acids spontaneously break down in a time dependent manner to tions to development and to gene expression at later stages (Zander
release ammonium, which subsequently affects cell physiology. This phe- et al., 2006). There is evidence that the preimplantation embryo may
nomenon is well-characterized in the tissue culture literature, and am- have an increasing ability to adapt or become tolerant to ammonium
monium generated from amino acids represents a significant problem as development progresses beyond compaction (Zander et al., 2006).
for the maintenance of somatic cell function and the maintenance of Edwards et al. (1998b) identified that the preimplantation embryo’s
lines (Newland et al., 1990; Schneider et al., 1996). Hence this is not a ability to regulate pH stress increases significantly post-compaction. Pre-
phenomenon unique or peculiar to mammalian embryo culture. For compaction embryos exposed to a weak acid showed a decrease in intra-
example, hybridoma cells utilize glutamine as their primary nitrogen cellular pH, while post-compaction embryos were able to maintain pH at
source and release ammonium as a metabolic waste product. This am- the physiological level (Edwards et al., 1998b). Ammonium is a weak acid
monium quickly accumulates to toxic levels in hybridoma culture and would therefore significantly inhibit the ability of a pre-compaction
media, thereby severely reducing monoclonal antibody production embryo to regulate intracellular pH. Consequently, this inability to main-
(Ozturk and Palsson, 1991). Gardner and Lane (1993) were first to iden- tain normal levels of intracellular pH could be associated with reduced
tify the presence of increasing quantities of ammonium over time in developmental competence and viability (Lane and Gardner, 2003;
embryo culture media and to reveal that the developing embryo also pro- Zander et al., 2006).
duced measurable levels of ammonium from the transamination of amino Although ammonium has been shown to be embryo toxic, few studies
acids. It was subsequently determined that the generation of ammonium have investigated the mechanism(s) by which the early embryo can regu-
within the culture medium had adverse effects on mammalian embryo late ammonium. Alanine and glutamine have been proposed as having a
development, physiology and viability (Gardner and Lane, 1993; Lane role in preventing the build-up of ammonium ions in culture medium.
and Gardner, 1994, 1997, 2003; McEvoy et al., 1999; Sinawat, 2001; Pyruvate, after transamination to alanine, has been suggested as an am-
Sinawat et al., 2003; Zander et al., 2006; Rooke et al., 2007). monium sink (Donnay et al., 1999; Orsi and Leese, 2004). However,
In embryo culture media ammonium accumulates to levels from alanine production did not increase when bovine embryos were
50 mM (Gilbert et al., 2012) to 300 mM (Gardner and Lane, 1993) de- exposed to increasing concentrations of ammonium (Orsi and Leese,
pending on the concentration of amino acids present and duration of 2004). Glutamate metabolism, catalysed by glutamine synthetase,
embryo culture. In the human, ammonium has been shown to induce could remove free ammonium ions through conversion to glutamine
not only a negative effect on blastocyst formation (Virant-Klun et al., (Orsi and Leese, 2004). Glutamine production at the blastocyst stage
2006), but also to alter metabolic activity and subsequent gene expres- has been reported in cattle (Orsi and Leese, 2004; Sturmey et al.,
sion of the human embryo (Gardner et al., 2013), with the former 2009), mice (Lamb and Leese, 1994) and pigs (Humpherson et al.,
being affected by as little as 75 mM. Hence, there is a trade-off 2005). Interestingly, there have also been reports on the varying effect
between the highly beneficial effects of amino acids and the detrimental of ammonium on preimplantation embryo development when different
8 Wale and Gardner
oxygen concentrations were used by different investigators (Lane and independently altered overall amino acid turnover. Together, 5%
Gardner, 1994; Biggers et al., 2004). oxygen and ammonium promoted glutamine production, whereas in
Wale and Gardner (2013) therefore considered the effect of ammo- the presence of atmospheric oxygen and ammonium, glutamine was
nium on the physiology of the embryo under different oxygen concentra- consumed (Fig. 2b). These data reveal that both ammonium and
tions (as a second source of stress in a culture system). Glutamine and oxygen affect the amino acid utilization by the developing embryo and
alanine synthesis were investigated as possible pathways used by the pre- that atmospheric oxygen appears to have a greater detrimental effect.
implantation embryo in ammonium sequestration as well as the effect of Consequently, mouse blastocysts can alleviate ammonium stress by its
oxygen on the regulation of these pathways. Amino acid utilization by transamination to both glutamine and alanine, but only under physio-
blastocysts was determined after culture from the post-compaction logical oxygen conditions (Wale and Gardner, 2013). Hence when stres-
stage with 0, 150 and 300 mM ammonium (in either 5 or 20% oxygen), ses collide the outlook for the developing embryo can be bleak.
and with or without 500 mM L-methionine sulfoximine (MSO), an inhibi- Importantly, these newly identified interactions between ammonium
tor of glutamine synthetase. In the presence of MSO, ammonium pro- and oxygen resolve an apparent paradox in the literature with regards to
duction was significantly increased, glutamate was no longer consumed differing results on the effect of ammonium exposure (either continuous-
and glutamine formation decreased (Fig. 2a). Ammonium and oxygen ly in medium containing Eagle’s concentration of amino acids (Eagle,
1959) or in medium supplemented with ammonium chloride) on
mouse embryo and fetal development. Lane and Gardner (1994)
reported a 20% incidence of exencephaly following culture of mouse
embryos for 96 h from Day 0.5 in atmospheric oxygen and in a non-
renewed medium containing amino acids and 1 mM glutamine. When
embryos were cultured with exogenous ammonium in atmospheric
oxygen but for less time (either 72 h from Day 0.5 with 300 mM or
48 h from Day 1.5 with 600 mM), Sinawat et al. (2003) reported exence-
phaly at a rate of 4.5%. The interaction of ammonium and atmospheric
oxygen could therefore be associated with the reported high frequency
of exencephaly cases (Lane and Gardner, 1994). In support of this,
Biggers et al. (2004) employed 5% oxygen and reported a 1% incidence
of exencephaly when embryos were cultured in physiological oxygen in
non-renewed medium containing amino acids at half Eagle’s concentra-
tion and using glycyl-glutamine. These results highlight the importance of
understanding how specific culture conditions (e.g. media composition,
length of exposure, developmental stage to which treatment is applied
and the concentrations of oxygen employed) interact and impact on
the physiology of the preimplantation embryo, resulting in different ex-
perimental outcomes in terms of embryo and fetal normality. Notably,
experimental conditions need to be precisely reported and replicated
if data are to be correctly interpreted.
Of clinical significance, ammonium-induced changes have also been
observed in human IVF. For example, when Dumoulin et al. (2010) in-
vestigated the effects of culturing human embryos in two different
culture media under physiological oxygen, one medium contained free
glutamine and the second contained alanyl-glutamine (a heat-stable
Figure 2 (a)Effect of L-methionine sulfoximine (an inhibitor of glu- form of glutamine). Embryo transfer primarily occurred on Day 2
tamine synthetase) or ammonium (150 and 300 mM) on glutamine turn-
during which free glutamine would break down to produce up to
over of mouse blastocysts, with all cultures performed under 5%
150 mM ammonium in the culture medium, while the alanyl-glutamine
oxygen. Notches represent the confidence interval of the median, the
depth of the box represents the interquartile range (50% of the data), would produce ,10 mM ammonium (Lane and Gardner, 2003). There-
and whiskers represent the 5 and 95% quartiles. The line across the fore, data from the Dumoulin et al. (2010) study may plausibly be inter-
box is the median uptake or release. Significantly different from preted as an investigation into the effect of exposure to different
embryos incubated in control medium: **P , 0.01. Data from Wale ammonium concentrations during in vitro culture of human embryos.
and Gardner (2013). (b) Effect of oxygen with and without 150 mM am- Indeed, Gardner et al. (2013) determined that 150 mM of ammonium
monium on glutamine turnover of mouse blastocysts. Notches re- has a negative impact on the metabolism of cleavage stage human
present the confidence interval of the median, the depth of the box embryos. Despite the limited numbers of children born, Dumoulin
represents the interquartile range (50% of the data), and whiskers re- et al. (2010) reported both lower pregnancy outcomes and reduced
present the 5 and 95% quartiles. The line across the box is the median
birthweight following culture of human embryos in a medium with free
uptake or release. Positive values reflect glutamine production, negative
glutamine, indicating that ammonium not only delays development in
values reflect consumption. Significantly different from embryos incu-
vitro, but could affect the birthweight of live born singletons (Zandstra
bated in control medium: **P , 0.01. Data from Wale and Gardner
(2013). et al., 2015). It is important to note that the Dumoulin study was a com-
parison of non-equivalent culture media and there were also differences
in parental phenotype of the study groups which could therefore influ- Avoiding the negative effects of poor air quality requires an under-
ence the outcome in this study, i.e. the parents in the alanyl-glutamine standing of how toxicants can infiltrate the laboratory, the incubator,
group were heavier, and heavier parents have heavier children. De Vos and ultimately the culture media. With this in mind, careful consideration
et al. (2015) compared singleton live births resulting from singleton preg- should be given to the site and location of human IVF laboratories as there
nancies sourced from two different sequential culture media, which both is potential for VOCs and CACs to significantly affect the developing
utilize a stable dipeptide source of glutamine. In this large (total of 2098 embryo (Cohen et al., 1997; Hall et al., 1998; Khoudja et al., 2013) and
births) retrospective study no significance differences in mean singleton appropriate design of the laboratory is important (Boone et al., 2010).
birthweight were observed (De Vos et al., 2015) giving credence to the The use of low odour specialized paints and the avoidance of sealants
proposition that the difference in outcomes described by Dumoulin’s and toxic glues is also recommended (Cutting et al., 2004; Cohen
group may therefore be attributed to a chronic exposure of embryos et al., 2012). Positive pressure airflow in the laboratory coupled with
to ammonium and/or differences in parental weights. the use of air purification systems (Khoudja et al., 2013) and appropriate
Of physiological significance, the observation of ammonium-induced in-line filters for incubators (Merton et al., 2007) will help to minimize the
changes to the preimplantation embryo is not limited to studies on level of airborne contaminants and improved outcomes. Pragmatically,
in vitro culture. The supply of high levels of protein or urea (2.5%) during the use of appropriate in-line air filtration together with certified gas cylin-
peri-conception increases levels of ammonium in plasma (150 mM) ders, and suitable bench-top incubators, means it is feasible to effectively
and the reproductive tract (McEvoy et al., 1997; Gardner et al., 2004), isolate gametes and embryos from the gaseous environment of the
thus creating an environment that is disruptive to embryo development laboratory.
in vivo. In mouse and livestock models a high maternal protein diet during VOCs can be emitted by various sources within the laboratory, such as
the peri-conception period affects fertilization (Blanchard et al., 1990), consumables, and a VOC meter should be used to establish off-gassing
embryo development (Sinclair et al., 2000; Powell et al., 2006) and time for consumables (including packaging) within the laboratory.
quality (Gardner et al., 2004). Additionally, altered preimplantation de- Many laboratories ensure the off-gassing of their culture wares in a sep-
velopment and subsequent expression of type II insulin-like growth arate laboratory to the clinical IVF laboratory, where dishes and tubes can
factor receptor (IGF2R), an imprinted gene, can be affected by high ma- be placed in a laminar flow hood overnight to facilitate the safe emission
ternal dietary nitrogen levels (Powell et al., 2006). Given the strength of of VOCs. Packaging, therefore, has a major impact on the immediate use-
data from controlled in vitro studies, combined with data from dietary ability of such plastics, with gas-permeable packaging allowing for use
interventions, it is plausible to suggest that elevated ammonium may ‘straight out of the box’. Further, laboratory personnel can also introduce
have negative influences in vivo, however the extent to which the pericon- VOCs in the form of perfumes and deodorants, hence discretion is
ceptual environment is buffered against this is unknown. required. Finally, given the concept of when stresses collide in the labora-
In closing this section, it is important to acknowledge that there are tory, the use of a reduced oxygen environment should help to reduce
reports that ammonium does not affect embryo development or physi- possible toxic effects of VOCs.
ology (Menezo and Guerin, 2007). However, in light of data from both
in vivo and in vitro studies, and data from somatic cell cultures, it is hard
to support the argument that ammonium is something that one can Albumin; blood versus recombinant
simply disregard with regards to the development of the mammalian The early years of human IVF were characterized by the inclusion of
preimplantation embryo. patient or fetal cord serum in the culture media used. This was done ir-
respective of whether the medium was a simple salt solution, such as
human tubal fluid medium (Quinn et al., 1985), or a complex tissue
Volatile organics culture medium, such as Ham’s F-10 (De Kretzer et al., 1973; Leung
Poor laboratory air quality is a recognized hazard to the culture of human et al., 1984; Zamboni et al., 1986). Although the use of sera facilitated
gametes and embryos (Legro et al., 2010; Perin et al., 2010). However, early embryo development during a time of sub-optimal laboratory con-
the cumulative data on volatile organic compounds (VOCs) and chemical ditions, it introduced many unknown variables into the culture system
airborne contaminants (CACs) are rather anecdotal, with the ability to (due to different diets of the patients and their physiological status at
investigate potential physiological effects limited by design. As such, the time of collection), making standardization of culture impossible
the concentrations of the contaminants, such as acrolein or nitrogen (Gardner and Lane, 2007). Furthermore, over the years there have
dioxide, which result in gamete or embryo toxicity, remain poorly been growing concerns about the use of whole sera for embryo
defined, but their effects on other tissues have been documented: ex- culture given its documented adverse effects on the embryos of labora-
posure to the VOC 1-octen-3-ol and its enantiomers caused a dose- tory and domestic animals. In mice, the addition of serum is associated
dependent decline in cell viability and cytotoxicity of human stem with altered expression of growth-related imprinted genes, which culmi-
cell line H1, as determined by a MTS (3-(4,5-dimethylthiazol-2-yl)-5- nates in aberrant fetal development (Khosla et al., 2001). In sheep and
(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay to cattle the inclusion of serum (human or autologous) induces premature
establish cell toxicity and a ‘live and dead’ stain (Inamdar et al., 2012). cavitation of morulae (Walker et al., 1992) and damages the intracellular
Further, exposure of murine bone marrow stromal cells to oct-1-en-3-ol integrity of embryos, disrupting mitochondrial membrane organization,
and a 2nd VOC (E)-2-octenal caused a shift to unsaturated fatty acids and thereby compromising metabolism (Gardner, 1994; Thompson
and lower cholesterol levels in the membrane, which is an indication of et al., 1995). Of greater concern, the presence of serum induces the de-
increased membrane fluidity, and changes to the cell membrane are velopment of abnormally large offspring, whereby the lambs or calves can
known to contribute to the breakdown of normal cell function and possibly be up to 50% greater than normal birthweight (Behboodi et al., 1995;
lead to death (Hokeness et al., 2014). Thompson et al., 1995; Sinclair et al., 1999; Rooke et al., 2007).
10 Wale and Gardner
Consequently, the move from whole serum to serum albumin Physical factors
reduced a significant amount of the variability, and was shown to be
equally, or more, effective as a protein source (Laverge et al., 1997).
However, with serum albumin significant lot to lot differences were
Temperature and pH
evident with regard to their ability to support embryo development Although at first glance ensuring constant temperature and pH would
(Kane, 1983; Batt et al., 1991). Further, the use of serum albumin appear to be straightforward, it remains a technically demanding chal-
carries with it the finite risks of disease transmission associated with lenge to minimize variations of both parameters around their set
the use of blood-derived products. From a chemical perspective it is points. The importance of temperature regulation is of greatest signifi-
evident that serum albumin brings to the culture medium far more cance for the oocyte, followed by the cleavage stage embryo, with
than bound fatty acids and other metabolites, such as citrate (Kane, increased thermotolerance increasing after compaction. If the tempera-
1983) and steroids. ture goes over 378C by a couple of degrees, even for just a transient time
A variation on the use of human serum albumin (HSA) in human of 20 min, then in the metaphase II oocyte the spindle starts to disassem-
embryo culture was considered by Pool and Martin (1994), who used ble and does not always completely reconstitute when the temperature
a plasma protein fraction, Plasmatein, characterized by a high globulin is brought back to 378C (Sun et al., 2004). The embryo responds to heat
content. This concept was developed further by Weathersbee et al. by expression of stress response genes, associated with loss of develop-
(1995) who compared the efficacy of HSA to a synthetic serum substi- mental competence (Hansen, 2007), whilst an earlier study suggests that
tute (SSS), characterized by high levels (16%) of both a and b globulins. a fall in temperature during oocyte handling is inconsequential (Bernard
Using this approach accelerated growth of human embryos at 38 h post et al., 1992) when fertilization and culture for a further 24 h are the mea-
insemination was reported. Subsequently, several forms of enhanced sured end-points. More recent studies have reported a decrease in tem-
albumin have become commercially available. In a randomized trial perature, whether drastic (Zenzes et al., 2001) or mild (Wang et al.,
Meintjes et al. (2009c) observed an 11% increase in pregnancy rate 2002), has the potential to affect the stability of the meiotic spindle of
when SSS was used in a blastocyst culture system as opposed to HSA the oocyte. The reported consequence of this alteration to the
alone (Meintjes et al., 2009c). Anecdotal evidence indicates that oocyte’s meiotic spindle is reduced fertilization rates (Wang et al.,
certain batches of such products work well, whereas others are 2002), delayed embryo development (Wang et al., 2001) and decreased
perhaps less effective than serum albumin alone, indicating that there clinical pregnancy rates (Wang et al., 2002).
may be factors in such albumin preparations other than globulins that It would therefore seem prudent to take rigorous steps to ensure that
have embryo trophic effects. all warming stages undergo daily monitoring using appropriately accurate
Of note, two recent and independent analyses have determined that and calibrated equipment. Furthermore, stages on microscopes should
commercially used serum albumin preparations used in human IVF not necessarily be set to 378C. Rather the temperature to which the
contain an abundance of non-declared proteins as well as transition gametes and embryos are exposed to in the dish depends on the
metals (Dyrlund et al., 2014; Morbeck et al., 2014). Further, the process- type/design of the dish, and whether there is a lip on the base, which
ing of albumin can lead to the introduction of known chemicals, such as will create an air pocket, and affects the thermal conductivity of the
octanoic acid, which has been shown to be detrimental to embryo devel- dish itself. Consequently, the temperature of warming stages needs to
opment (Leonard et al., 2013). From our own analyses we have also be calibrated within the culture medium inside the dish itself. This fre-
determined high levels of albumin-bound compounds in culture quently translates to a temperature of .388C on the warming stage.
medium, such as ethanol and caprylate, which are used in the extraction As will be discussed with regards to pH, a possible solution to variations
and stabilization of albumin, respectively (Sheedy and Gardner, unpub- in temperature is to work within an isolette/humidified chamber, which
lished observations). Additionally, there are now concerns that serum can maintain air temperature (and consequently any equipment housed
albumin, added as the protein supplement, is the source of detectable within the heated chamber) close to 378C, therefore preventing equip-
levels of Di(2-ethylhexyl)phthalate and mono(2-ethylhexyl)phthalate, ment within from acting as heat sinks.
as well as polybrominated diphenyl ethers in human embryo culture More recently it was proposed that working at a temperature of 368C
media (Takatori et al., 2012; Akutsu et al., 2013). Such data infer that may mimic more closely the temperature of the female reproductive
the use, and/or preparation, of serum albumin in human IVF warrants tract (Bahat et al., 2005), and that consequently a lower temperature
renewed consideration. To this end, recombinant human albumin has better supports a ‘quieter’ embryo development (Leese et al., 2008).
been shown to be an effective replacement for serum-derived albumin However, in a RCT, there was no improvement in human embryo devel-
in IVF and embryo culture (Bavister et al., 2003; Lane et al., 2003) and opment or clinical outcomes when 368C was used compared with the
its clinical efficacy validated (Bungum et al., 2002). Given the growing con- standard of 378C (Hong et al., 2014). To date, therefore, the data indi-
cerns surrounding the safety of human IVF, it would appear that a move cate that maintaining gametes and embryos at 378C is advisable and ef-
towards recombinant albumin to minimize risk and to increase consist- fectual.
ency of function, much like the move from urinary to recombinant gona- In order to regulate the pH of fluids, when working outside of the body
dotrophins, is timely. When the use of recombinant albumin was first it is typical to employ a bicarbonate/carbon dioxide buffering system. By
considered, the cost of the recombinant protein was almost one manipulating the concentration of carbon dioxide and the concentration
hundred times that of serum-derived albumin. However, over the past of bicarbonate, typically present as the sodium salt, it is readily feasible to
two decades the costs for such recombinant materials has decreased create the required pH. This approach has the advantage that it is easy to
considerably, making the inclusion of recombinant albumin in human manipulate the set pH of the culture medium by changing either the
IVF and embryo culture media a feasible proposition. Hence it is time bicarbonate or carbon dioxide concentration. However, in reality it
to re-evaluate its use clinically. takes careful attention to detail with regards to calibration of the
incubator and pH measuring device, in order to ensure that the pH of Similarly, the addition of amino acids and protein to a medium will also
the culture medium inside the incubator is what one expects it to be. affect medium pH.
Calibrated digital carbon dioxide analysis units are preferred over a
liquid-based system such as Fyrite. Significantly, as the accuracy of Oil overlay
electronic-based gas analysis unit falls during its lifetime, one must regu-
Due to the small volumes of medium typically used for IVF and embryo
larly check the accuracy of these monitors against a known standard. The
culture (between 20 and 250 ml) an oil overlay is used to prevent
analysis of medium pH is best performed with a blood-gas analyser, or an
evaporation and hence stop the medium becoming hyperosmotic.
optical device inside the incubator. Standard pH meters can be used for
Although mammalian embryos can tolerate a wide range of osmolality
such measurements, but it is technically challenging given that the pH of
(200–350 mOsmol) (Gardner and Lane, 2007; Baltz, 2012), without
the media will rapidly increase outside of the incubator. Consequently,
an oil overlay the osmolality would rapidly become too high for the
when measuring pH it is important to snap the tube of test media
embryo to survive. The inclusion of such an overlay also provides stability
closed whilst inside the incubator prior to injection into a blood gas ana-
with regards to medium pH, reducing the magnitude of medium pH oscil-
lyser. If a pH probe is used then it needs to be at 378C to obtain a mean-
lations. Consequently, oil has a very important place in the IVF labora-
ingful reading, or alternatively use a probe with temperature adjustment.
tory. The oil typically used in embryo culture is mineral (also known as
An increase in the pH of the culture media can have a highly detrimen-
paraffin) oil. Mineral oils are derived through the production of petrol-
tal effect on oocyte and embryo physiology and development (reviewed
eum, and can be classified according to viscosity. Given its industrial
by Swain, 2012). Of note, even a transient exposure to acidified media
origin it is essential that the quality of oil used is ensured, which can be
can have ensuring effects on both fetal weight and length (Zander-Fox
achieved through an appropriate bioassay (Gardner et al., 2005). It is
et al., 2010). Therefore, although the human embryo possesses several
our experience that embryo development is enhanced at higher oil
intracellular mechanisms to regulate its internal pH (Phillips et al.,
viscosity (Gardner and Wale, unpublished observations). Oils are not
2000) care must be taken to minimize fluctuations of pH. An excellent,
chemically inert; rather oil readily removes hydrophobic compounds,
and pragmatic, means of doing this is to use isolettes capable of maintain-
such as steroids. Further, it has the capacity to oxidize over time, so it
ing a more stable medium pH through the provision of carbon dioxide gas
should be stored in a cool dark place. Alternatives to mineral oils
within the chamber. This approach has proven to be effective. If isolettes
include silicon, but there has been limited clinical validation of such
are not available and one needs to keep embryos out of an incubator,
reagents (Erbach et al., 1995; Van Soom et al., 2001). Oils also can
then the inclusion of either HEPES and/or MOPS can serve to maintain
come with many impurities, some of which have been shown to be
a constant pH for embryo manipulations (Gardner and Lane, 2007; Swain
embryo toxic (reviewed by Morbeck and Leonard, 2012). Consequently,
and Pool, 2009). Whilst numerous studies indicate HEPES is able to
protocols have been developed to wash oils, either with medium or with a
support oocyte maturation, fertilization and embryo development at
solution of chelators such as EDTA, in an attempt to improve the quality
room atmosphere, only short exposure to HEPES and/or MOPS is
of the oil used in the IVF lab. Whilst Morbeck et al. (2010) provide clear
recommended as long-term exposure has been reported to result in
evidence (including a thorough protocol) that washing reduces the tox-
oocyte degeneration, lower fertilization rates, and/or compromised
icity of mineral oil, the washing of oils, although it appears an attractive
blastocyst formation (reviewed by Will et al., 2011). Similar to data on
concept, warrants a considerable amount of time and resources. Alterna-
the effects of temperature, the effects of pH drifts appear greater on
tively, if oil can pass a suitable bioassay, such as a 1-cell mouse embryo
the oocyte than the cleavage stage embryo (Lane and Gardner, 2000),
(Hughes et al., 2010), in a reduced volume (2–5 ml) in order to increase
while at compaction (and the formation of a transporting epithelium)
surface area to volume ratio in order to increase sensitivity, then washing
the embryo develops greater ability to maintain intracellular pH (Edwards
should not alter its efficacy.
et al., 1998b). Furthermore, temperature is a key affecter of intracellular
pH (Lane, 2001). Consequently, one needs to address both parameters
with equal gravitas. Incubation volume/embryo density
The significant impact that the percentage of carbon dioxide has on Human embryos are cultured in volumes ranging from a few microliters
maintaining medium pH is sometimes overlooked, and too many up to 1 ml (typically when using test tubes or 4-well plates, Bolton et al.,
patients’ embryos are all too commonly cultured in the same incubator, 2014). Further, with the advent of both preimplantation genetic
resulting in multiple door openings, thereby compromising the level of screening (PGS) and time-lapse microscopy, there has been a move to
carbon dioxide and consequently medium pH. Therefore, it is recom- single embryo culture. Interestingly, animal studies have revealed that
mended to ensure sufficient chambers are made available to safely culturing embryos in smaller volumes and/or groups significantly
accommodate the number of cycles performed. Hence working increases blastocyst cell number and elevates embryo viability (Wiley
quickly outside the incubator, together with careful attention to safe et al., 1986; Paria and Dey, 1990; Lane and Gardner, 1992; Doherty
and gentle pipetting of embryos, is essential, and needs to be instilled et al., 1997; Donnay et al., 1997). However, Spyropoulou et al. (1999)
and reinforced as a fundamental of successful embryo culture. If standard could not replicate the positive effects of group culture with human
tissue culture type incubators are selected, then it is best to ensure that embryos, although it should be noted that embryos were only cultured
they have an infra-red CO2 sensor, rather than a thermocouple-based to Day 2. In regards to blastocyst development, Rijnders and Jansen
sensor which is humidity dependent and therefore much slower to (1999) cultured human embryos from day 3 to the blastocyst stage indi-
react to changes in gas composition and has a slower recovery time vidually, or grouped in large and small volumes, and found no significant
(Swain, 2014). Factors such as altitude will affect the partial pressure of different in blastulation rates between the four groups. They concluded
carbon dioxide in the medium, and hence it is paramount to measure that whilst all of the four treatments yielded similar results individual
pH rather than simply relying on the carbon dioxide measurement. culture in a small volume allowed for direct and individual assessment
12
Table 1: Summary table of key human embryo culture studies of individual and/or group culture.
Paper Summary Start End Culture medium Protein source Individual Group Oxygen Outcome
conditions conditions concentration
..........................................................................................................................................................................................................................................................
Moessner and Individual culture versus group Day 1 Day 2 Modified Ham’s F10 15% human serum 1 ml 2 –5 embryos 20% Significantly greater MCN
Dodson (1995) culture, same volume per 1 ml from group culture, positive
correlation between size of
the co-culture groups and cell
number
Almagor et al. Individual culture versus group Day 1 Day 2 Ham’s F10 10% serum – cord 700 ml 3 –6 embryos 20% Increased pregnancy and
(1996) culture, same volume blood per 700 ml implantation rates from group
culture
Spyropoulou Individual culture versus group Day 1 Day 2 Earle’s balanced salt Synthetic serum 20 ml 3 –5 embryos 20% No statistical significance
et al. (1999) culture, same volume solutions containing replacement per 20 ml difference
0.11 mg/ml pyruvate (0.001%) + 1% (v/v)
human serum albumin
Rijnders and From Day 3, four conditions: Day 3 Day 5 Mixture of Earle’s and Pasteurized plasma (a) Small (c) 8– 12 20% No statistical significance
Jansen (1999) single culture in small volume Ham’s F10, without solution (8.7%) volume (5 ml) embryos (5 ml difference
or group culture in large hypoxanthine and (b) Large per embryo)
volume and vice versa thymidine volume (d) 160 ml total
(160 ml) volume
Rebollar– Individual culture versus group Day 1 Day 3 Cook SIVF medium Not specified 15 ml 3 –5 embryos Not specified Similar pregnancy and
Lazaro and culture through cleavage stage, (culture per 15 ml implantation rates
Matson (2010) all embryos cultured continued to Group culture (up to Day 3)
individually from day 3 to Day 5) increased blastocysts utilized
blastocyst stage
Ebner et al. Individual culture (with and Day 1 Day 5 EmbryoAssist – Not specified 30 ml (OWI) 3 –5 embryos Not specified Group culture was superior
(2010) without contact– using Medicult 4 embryos per per 30 ml for compaction and
specially designed culture 30 ml (CW) (OWG) blastulation as well as overall
dishes that allow for individual blastocyst quality
identification of embryos) and Trend to higher live birth rate
group culture with group culture
Tao et al. (2013) Group culture from Day 3 Day 3 Day 5 Vitrolife G1 and G2, Not specified Not applicable 2 –5 embryos 5% Group culture of good
embryos in which good or poor v5) per droplet 50 ml embryos from day 3
quality embryos were significantly promoted
separately grouped blastocyst development
Restelli et al. Group culture with embryos Day 1 Day 5 Sage Quinn’s Not specified Not applicable ‘Random Not specified Similar results in terms of
(2014) either randomly grouped or sequential media Group’ 3-4 blastulation rate and the
grouped based on pronuclear embryos per random grouping of zygotes
pattern 20 ml improved pregnancy and
Wale and Gardner

‘Definite implantation rates in
Group’ upto 4 IVF-cycles
embryos per
20 ml
of embryo morphology and therefore was preferable. Subsequently,
formation rate observed from
observed during day 3 to day 5

Significantly higher blastocyst
Ebner et al. (2010) performed a much larger prospective study compar-
embryos cultured in 7 ml
ing single and grouped culture using specially designed culture dishes that
drops, with differences

allowed for the individual identification of embryos. Group culture was
superior for both compaction and blastulation as well as overall blasto-
cyst quality, with Ebner et al. (2010) concluding that culture volume
should be reduced and embryo density increased. Table I lists studies
phase
on human embryo group versus individual culture, including key informa-
tion such as duration of culture, embryo density and the outcome
observed.
This supportive effect of group culture is plausibly the result of the
Not specified
preimplantation mammalian embryo producing a factor(s) which can

stimulate development. Several factors have been identified which
have bioactivity in vitro (reviewed by Thouas et al., 2015). Secreted
factors, such as platelet activation factor (PAF) and ubiquitin, have
Incorporated in culture 35 ml vs 15 ml Not applicable
been identified and in some cases quantified in culture medium condi-

tioned by human embryos; PAF has been identified in culture medium
conditioned by human, murine, hamster, rabbit and sheep embryos
with roles in cell cycle progression, embryo metabolism and viability
(reviewed by O’Neill, 2005). Of interest, in relation to the human
or 35 ml vs 7 ml
embryo a positive correlation between clinical pregnancy and the level

of PAF in culture medium was reported by Roudebush et al. (2002).
Ubiquitin is secreted by human embryos and is detectable in culture
medium, and has been correlated with blastocyst development
(Katz-Jaffe et al., 2006). Other possible factors include insulin-like
growth factor 1 (IGF1) and IGF2, platelet-derived growth factor alpha,
basic fibroblast growth factor, transforming growth factor beta and inter-
feron, as the appropriate mRNA for these growth factors are expressed
MNC, mean cell number; OWI, outer well individual; CW, centre well; OWG, outer well group; 2PN, pronucleate oocyte.
medium
by the bovine preimplantation embryo (Watson et al., 1992). However,

the physical presence of several of these growth factors in embryo
culture media has yet to be demonstrated.
Perhaps not surprisingly paracrine factors appear to be common
Advantage protein
amongst mammalian species. Spindler et al. (2006) demonstrated that

Sage Quinn’s
plus medium
companion mouse and cattle embryos conferred a benefit to singleton

cat embryos, whilst the number (and quality) of companions embryos
necessary to grant an advantage may be species dependent (Spindler
et al., 2006). Other groups have also demonstrated that paracrine
factors have a limited effective range. Stokes et al. (2005) used a novel
method to investigate the development of porcine embryos, which
Day 5
allowed distance between adjacent embryos to be varied by securing

the embryos to the base of a petri dish coated with Cell-Tak. The devel-
Sibling 2PNs were cultured in Day 1
opment of individual porcine embryos to the blastocyst stage was

optimal when they were cultured 81 –160 mm apart and as the distance
35 mL or 7 mL drops. Two end
between the embryos was increased, blastocyst rates declined signifi-

either 35 mL or 15 mL, or in
points were analysed; day 3
cantly, reaching zero beyond 640 mm (Stokes et al., 2005). Using the
(before medium change)
same novel method, Gopichandran and Leese (2006) investigated the

development of bovine embryos. Interestingly, similar to the porcine
embryos, the optimal bovine blastocyst formation rate occurred when
embryos were cultured 165 mm apart, which lends further support to
and day 5
the notion that paracrine factors are common amongst mammalian

species.
Whilst the identity of these factors remains largely unknown, their sig-
nificance is being considered through the morphokinetic analysis of
Minasi et al.
embryo development. Wydooghe et al. (2014) used time-lapse analysis

(2015)
to study the influence of ‘neighbours’ on a bovine embryo’s potential to

reach the blastocyst stage. They demonstrated that it is not necessarily
the number of neighbouring embryos that impede an embryo’s potential
14 Wale and Gardner
to reach the blastocyst stage but rather the developmental stage of the The decision to culture embryos individually or in groups is typically a
neighbours. An embryo’s development was evaluated at 45 h post in- decision based on historical laboratory driven protocols, convenience
semination (hpi) with embryos which had proceeded through the third and/or the need to collect data on individual embryos, as in the case of
cleavage division (5 –8 cells) classified as ‘fast’ and embryos which PGS. Habitually, embryo density is not considered a physical factor that
were between 2 and 4 cells (second cleavage division) categorized as may affect mammalian embryo culture, and its importance in the practice
‘slow’. Time-lapse analysis revealed that a bovine embryo’s development of assisted human reproduction is often overlooked. Embryo density can
after 45 hpi can positively affect the outcome of its neighbours, with a be controlled and, as such, it can be utilized as a simple, yet effective
markedly higher percentage of ‘slow’ embryos developing into blastocysts tool to improve in vitro development of human embryos (Reed, 2012).
by 192 hpi if they had been surrounded by many embryos that had also
developed into blastocysts by 168 or 192 hpi when compared with Pipetting induced shear stress
‘slow’ embryos cultured in isolation (Wydooghe et al., 2014). These In vivo, embryos do not experience acute shear stress; such stress is
results suggest that production of factors may be associated with induced by high-sustained velocity through too vigorous handing.
embryo quality, and warrant evaluation clinically. Up-regulation of phosphorylated mitogen-activated protein kinase
Of interest, the work by Tao et al. (2013) suggests that embryo- (MAPK)8/9 is a marker of MAPK8/9 activation in response to stress
to-embryo communication may not always be positive, with poor-quality (Xie et al., 2006). Pipetting embryos can result in the up-regulation of
embryos possibly exerting a negative influence on development of good- phosphorylated MAPK8/9 in a dose-dependent manner (Xie et al.,
quality embryos in group culture. Caution also needs to be exercised 2007). In comparison, when measured in in vivo-derived embryos,
when considering what volume and configuration should be used for from embryonic day (E)1.5 to E4.5, phosphorylated MAPK8/9 was
culture of embryos. When Dumoulin et al. (2010) evaluated two differ- expressed at low levels (Xie et al., 2007). After 24 h embryos that had
ent media, individual embryos were cultured in only 5 ml drops of previously been pipetted sufficiently to induce phosphorylated
medium under oil for 2–3 days after egg retrieval (embryo transfer oc- MAPK8/9 displayed the same number of cells as untreated, which sug-
curred on Day 2 or 3). The choice to employ such low volumes could gests that rapid phosphorylation of MAPK8/9 as a result of transient
have further contributed to the ammonium build in both groups, shear stress does not mediate long-term negative effects (Xie et al.,
coupled with the additional ammonium from the medium containing 2007). However, it is not known if multiple handling events could
free glutamine. impact biological outcomes (Xie et al., 2007). These data suggest that
Figure 3 Synergistic effects of two stresses in an embryo culture system. In the presence of a single stress, embryo physiology can be compromised.
When two stresses combine in the system then further, and potentially synergistic, negative effects become apparent. The example depicted is ammonium
accumulation in the presence of atmospheric oxygen (Wale and Gardner, 2013). A further example is the exposure of embryos to light while at room tem-
perature (Fischer et al., 1988).
Figure 4 An overview of key chemical and physical factors that affect mammalian embryo development in vitro; red represents chemical factors and blue
represents physical factors. QC, quality control; QA, quality assurance; VOC, volatile organic compounds.
embryo handling, a necessary aspect of human ART, should be per- culture without medium replacement is associated with the accumula-
formed with care and kept to a minimum. Coincidently, the emergent tion of the end products of metabolism and the breakdown products
time-lapse technology offers the ability to reduce the number of handling of labile components, as well as any toxins coming off the culture dish.
events required whereas the introduction of routine embryo biopsy for Evidently, we are faced with a ‘Catch 22’ with regards to medium
genetic testing increases the required handling events. renewal; to renew the medium allows for toxin removal and nutrient re-
plenishment in a stage-specific fashion, while to leave an embryo undis-
tributed minimizes trauma from handling and shifts in pH and
Static nature of culture temperature. Both approaches have been shown to work clinically.
Given that undisturbed culture will reduce the cellular trauma associated With the advent of time-lapse incubation systems, there is a growing
with shifts in temperature and pH as embryos are removed from the in- trend to leave embryos in the same well for 4 –5 days. Further, such un-
cubator, and with subsequent pipetting, it may appear at first glance that disturbed culture will also avoid possible dilution of embryo-derived
leaving embryos in the same drop of medium for extended culture is a autocrine factors. A potential scenario to accommodate the best of
logical means to culture human embryos. However, this approach both of the above approaches to culture would be to remove medium
creates a very artificial condition; a static environment. The reality is (say 75%) after 48 h from the time-lapse dish and add to the remaining
that the human embryo in vivo is exposed to a highly dynamic environ- medium a second phase medium designed to create a new and yet rela-
ment. Not only is the embryo moved constantly, but it is exposed to gra- tively undisturbed environment. This approach could also assist with the
dients of nutrients (Gardner et al., 1996) and to hormones, cytokines and retention of embryo-derived factors in the culture media. Ultimately,
growth factors at stage-specific times (Thouas et al., 2015). The embryo embryo culture may be performed in a continual flow system as advo-
itself is metabolically highly active, and hence is constantly changing its cated over 20 years ago by Gardner (1994), facilitated by the develop-
own environment in culture through its consumption of nutrients and ment of microfluidic devices (Swain et al., 2013), able to provide a
release of metabolites (Gardner, 2008). Consequently the formulation dynamic environment and facilitate the removal of toxins whilst facilitat-
of a 25 ml drop of culture medium at the commencement of culture is ing image collection and biomarker detection (Gardner et al., 2015).
far removed from that after 4 days of embryo culture. Furthermore,
the later stage embryo will create a phenomenon known as an unstirred
layer (Trimarchi et al., 2000), in which the embryo creates a gradient of Light
nutrients (particularly true in a column of fluid), which typically results in Within the female reproductive tract fertilization and embryo develop-
nutrient insufficiency at low substrate concentrations. Hence, continual ment will occur in the complete absence of light. In contrast, under
16 Wale and Gardner
Table II Embryo development in the laboratory is a balance of different stresses.
Dynamic environment Static environment

.............................................................................................................................................................................................
Alleviates ammonium accumulation and diminishes the impact of unstirred Ammonium and other embryo-derived metabolites accumulate, and the
layers embryo creates extended unstirred layers
Potential loss of paracrine factors derived from the cleavage stages Potential cumulative benefit of paracrine factors derived from the cleavage
stages
More pipetting; potential for pH and temperature shifts, and shear-stress Less pipetting; no pH or temperature drift during medium change over on Day 3,
during medium change over (although this could be overcome by replacing the and reduced potential for shear-stress activated factors
majority of the medium drop itself and not moving embryos)
Changing nutrient pool to mirror the gradients of nutrients to which the Constant level of nutrients with plausible metabolic consequences
embryo is exposed as it progresses through the oviduct to the uterus in vivo
Human embryos are currently cultured in either a dynamic environment such as that facilitated by sequential media, or in a static environment, through the use of a single medium for all
stages. The use of either system encounters some form of compromise, which are considered throughout the text. The influence of one or more factors in either culture system therefore
has the capacity to affect the efficacy of the type of media/environment used. Consequently, it is conceivable that optimal embryo development has yet to be achieved, and will plausibly
require a device (potentially one based on microfluidics) to create an environment where all stresses discussed can be alleviated.
Figure 5 Relative impact of chemical and physical stress on preimplantation embryo (from the fertilized oocyte to the blastocyst stage), representing the
stage-specific differences in the embryo’s response to stress. The fertilized oocyte is more sensitive than the cleavage stage embryo, which in turn is more
susceptible to stress than an embryo post-compaction. Of all stages, the blastocyst is least perturbed by such factors.
conventional laboratory conditions, gametes and embryos are exposed With regards to the oil used to overlay culture media droplets, studies
to light of various wavelengths, intensities and sources (both ambient and have shown that as a result of exposure to light, peroxides can form in a
during assessment under a microscope). Using hamster and porcine cascade reaction, the result of which is transfer of water soluble contami-
models, it has been shown that light can directly alter embryonic devel- nants into the culture drops (Otsuki et al., 2007, 2009). So it is important
opment (Oh et al., 2007; Li et al., 2014c), or indirectly via photo- to consider that whilst commercial oil products may arrive from the
oxidation of components in medium (Li et al., 2014c). Similar to previous manufacturer with a valid certificate of analysis, exposure to sunlight
stressors, such as oxygen and ammonium, Schumacher and Fischer and/or viable light during transportation or improper storage may
(1988) demonstrated that the pre-compaction stage embryo is more result in the defilement of the oil.
sensitive to direct light than the post-compaction stage. Furthermore ex-
posing Day 1 rabbit embryos to light for as little as 1 h resulted in
decreased cell proliferation as measured by the incorporation of thymi-
Conclusions
dine (Schumacher and Fischer, 1988). Light in the green spectrum The impact of culture media formulations on mammalian embryo devel-
(510 nm) has higher energy and should be avoided (Boone et al., opment has been extensively documented over the preceding five
2010). The advent of time-lapse systems which typically employ light decades. Improvements in the success of human ART can be attributed,
of long wavelength (625–635 nm using a red LED) and hence lower in no small way, to the continued efforts of laboratories around the world
energy, combined with very short exposure times (even when successive to optimize media formulations. However, what is evident is that media
images are added up over several days), should assist in reducing this are but one aspect of the embryo culture system, which comprises other
stress on the embryo (Chen et al., 2013). factors, both chemical and physical (Gardner and Lane, 2003). These
factors in isolation have been shown to have dramatic effects on embryo et al., 2015). Consequently, the impact of the factors described in this
physiology and viability. Furthermore, there can be cumulative effects of review on gametes and embryos may vary according to the status of
such stressors which act synergistically, as documented for the negative the patients, including their age and weight. Discussions arising from pub-
interactions between 20% oxygen and the ability of the preimplantation lications on the effects of human embryo culture media on resultant
mouse embryo to detoxify ammonium (Fig. 3). Similarly, when Fischer birthweights need to be tempered in light of the fact that so many vari-
et al. (1988) investigated the adverse effects of exposure to visible light ables can impact transfer outcome. Further studies on gamete quality
and room temperature in rabbit cleavage stage embryos and morulae, and the interactions of all factors associated with the embryo culture
it was determined that embryonic damage was detectable in both system will continue to provide a greater insight into the regulation of pre-
stages of development after 1 h exposure, but that the combined expos- implantation human embryo development, and the mechanisms asso-
ure to light and room temperature amplified the detrimental effects ciated with the programming of a healthy pregnancy and child.
(Fischer et al., 1988). Through the use of bench-top incubators and
emergent technologies (time-lapse) several of the described chemical
and physical factors can be addressed quite effectively, for example the Acknowledgements
use of 5% oxygen. An overview of several chemical and physical The authors thank Dr Rusty Pool, Rebecca Kelley and Lisa Lee for their
factors is represented in Fig. 4, and their presence and effects in different comments on the manuscript.
types of culture environment is considered in Table II.
It is also evident that there are clear stage-specific differences in the
embryo’s response to stress, and that the oocyte and cleavage stage Authors’ roles
embryo are far more vulnerable to their environment than the embryo
P.L.W. and D.K.G. contributed equally to the writing of this manuscript.
post-compaction (Fig. 5). As such, the success associated with blastocyst
transfer could reflect the stress associated with asynchronous transfer of
the cleavage stage embryo to the uterus, a setting which provides a dif- Funding
ferent environment to that which the cleavage stage requires (Barnes,
P.L.W. and D.K.G. were supported by the University of Melbourne.
2000; Walker et al., 2015). Nevertheless, some parties continue to
argue against the use of extended culture in human IVF, advocating the
transfer of the cleavage stage human embryo to the uterus (Brison Conflict of interest
et al., 2014). In the publication of Scherrer et al. (2012), apparently
healthy children conceived through IVF exhibited generalized vascular P.L.W. has no conflicts of interest. D.K.G. receives research funding from
dysfunction associated with ART, fuelling the discussion around labora- Vitrolife (AB).
tory effects. However, these children were derived from embryos trans-
ferred at either the pronucleate oocyte or 2- to 4-cell stage following
culture in medium lacking amino acids and grown in 1 ml of culture
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The Effects of Chemical and Physical Factors On Mammalian Embryo Culture and Their Importance For The Practice of Assisted Human Reproduction

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Human Reproduction Update, Vol.22, No.1 pp.

The effects of chemical and physical

*Correspondence address. E-mail: [email protected]

Wale and Gardner

of embryo morphology and therefore was preferable. Subsequently,

formation rate observed from

observed during day 3 to day 5

drops, with differences

preimplantation mammalian embryo producing a factor(s) which can

been identiﬁed and in some cases quantiﬁed in culture medium condi-

embryo a positive correlation between clinical pregnancy and the level

by the bovine preimplantation embryo (Watson et al., 1992). However,

amongst mammalian species. Spindler et al. (2006) demonstrated that

companion mouse and cattle embryos conferred a beneﬁt to singleton

allowed distance between adjacent embryos to be varied by securing

opment of individual porcine embryos to the blastocyst stage was

between the embryos was increased, blastocyst rates declined signiﬁ-

points were analysed; day 3

same novel method, Gopichandran and Leese (2006) investigated the

the notion that paracrine factors are common amongst mammalian

embryo development. Wydooghe et al. (2014) used time-lapse analysis

to study the inﬂuence of ‘neighbours’ on a bovine embryo’s potential to

Table II Embryo development in the laboratory is a balance of different stresses.

Dynamic environment Static environment

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