Reproductive BioMedicine Online (2010) 20, 453– 469

www.sciencedirect.com
www.rbmonline.com

REVIEW

Embryo culture: can we perform better than nature?

a,*,1
Gábor Vajta , Laura Rienzi c, Ana Cobo d, John Yovich a,b

a
Cairns Fertility Centre, Cairns, QLD 4870, Australia; b PIVET Medical Centre, 166-168 Cambridge Street,
Perth, WA 6007, Australia; c G.EN.E.R.A centre for Reproductive Medicine, Clinica Valle Giulia,
Rome, Italy; d IVI Universidad de Valencia Plaza de la Policı́a local, 3 46015 Valencia, Spain
* Corresponding author. E-mail address: gabor.vajta@cairnsfertility.com (G Vajta).
1 Current address: James Cook University, Cairns, QLD 4870, Australia.

Gábor Vajta obtained an MD degree (1976), a speciality (1979) and a PhD degree in human pathology (1988) in
Hungary, and a Doctor of Veterinary Sciences degree (1999) in Denmark. He is adjunct professor at the
University of Copenhagen, Denmark and James Cook University, Australia. He is author of more than 100
publications, and member of the Editorial Board of Cloning and Stem Cells.

Abstract Culture of preimplantation-stage embryos has always been a key element of laboratory embryology and has contributed
substantially to the success of many assisted reproduction procedures. During the past decade, its importance has increased as
extended in-vitro embryo culture and single blastocyst transfer have become indispensable parts of the approach to decreasing
the chance of multiple pregnancy while preserving the overall efficiency of the treatment. However, in spite of the scientific
and commercial challenge stimulating research worldwide to optimize embryo culture conditions, a consensus is missing even in
the basic principles, including composition and exchange of media, the required physical and biological environment and even
the temperature of incubation. This review attempts to summarize the controversies, demonstrate the fragility of some widely
accepted dogmas and generate an open-minded debate towards rapid and efficient optimization. New approaches expanding the
traditional frames of mammalian embryo culture are also discussed. Although some researchers suppose that the efficiency of
the presently applied in-vitro culture systems have already approached the biological limits, authors are confident that substantial
improvement may be achieved that may expand considerably the possibilities of future assisted reproduction in humans. RBMOnline
ª 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
KEYWORDS: blastocyst, human, incubation, in vitro, in vivo, stress

Introduction vanced in that period, at least compared with mammalian

embryology. Achievements in that particular decade in-
The first successful culture of mammalian embryos in vitro cluded harnessing nuclear fission for power generation as
was published in the middle of the last century (Whitten, well as an established military use in atomic and hydrogen
1956, 1957). It is probably worth mentioning that the overall bombs, the first space flights, development of synchrotrons,
level of physical and natural sciences was surprisingly ad- nuclear powered submarines and the cracking of the DNA

1472-6483/$ - see front matter ª 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.rbmo.2009.12.018
454 G Vajta et al.

code, whilst in biological science the production of a single Principles to reconsider

blastocyst in a Petri dish was still an unattainable dream for
most embryologists.
The first group of problems originates from the somatic cell
According to Bavister (2002), the major reason for this
analogue. All standard embryo culture procedures use so-
regrettable delay was that scientists did not realize the ex-
matic cell cultures as templates and the existing differences
treme temperature sensitivity of oocytes and embryos in
can only be regarded as slight modifications. The frames,
most mammalian species, particularly at those stages of
rules, tools and equipment of mammalian somatic cell cul-
meiosis involving delicate spindle formation. However, even
tures were established decades before the first successes
that eventual discovery (Shettles, 1953; Smith, 1951) did
in embryology and served as models for embryo culture.
not accelerate progress either. In spite of the impressive
The subsequent innovations on the somatic cell field
achievements in human IVF with millions of babies born
(including microbeads, smart substrates, microbioreactors,
worldwide, culture of mammalian embryos remains a
etc.; Mendes, 2008; Miller et al., 1989; Rahman et al.,
demanding task and is still performed using rather primitive
2009) had little or no effect on embryo culture in vitro. This
techniques and the advancement is slow.
unfortunate approach by embryologists completely disre-
To avoid fiascos, in practice most laboratories simply use
gards that the situation, morphology, function and regula-
their earliest albeit modestly successful routines, painstak-
tion of somatic cells in vivo are basically different from
ingly introducing any innovative procedures which require
those of preimplantation mammalian embryos.
many years, sometimes a decade or even more to develop.
These differences and their consequences are summa-
Controversially, even the few new procedures that gain
rized in the following points:
worldwide acceptance are typically implemented and ap-
plied without scientifically sound evidence. Consensus
among scientists is often lacking. Even the most widely ac- (i) Preimplantation embryos are not part of the maternal
cepted parameter, the application of the core temperature, body: they exist physically and biologically as largely
has been questioned recently and for good reasons (Leese independent living beings in an open tube. The physical
et al., 2008b). Due to these hurdles and other, not purely separation from the maternal body is reinforced by the
scientific factors, mammalian embryology may still be re- zona pellucida and becomes complete with the loss of
garded as a handicapped branch of science especially when cumulus cell–oocyte gap junctional communication.
compared with other fields, e.g. information technology, (ii) While somatic cells of mammals are surrounded by the
nanotechnology and molecular biology. extracellular fluid that is rich in nutrients, proteins
Nothing proves that the future cannot be brighter. It is a and other factors, preimplantation embryos are sur-
self-evident truism to refer to the in-vitro environment as rounded by an as yet undefined (but most probably
‘inappropriate’, ‘stressful’ and ‘evidently inferior’ com- very low) amount of oviductal and uterine fluid that
pared with the in-vivo counterpart. Ambitions are usually is more dilute, contains less resources and its phys-
restricted to narrowing the gap between the two systems, ico-chemical characteristics show spatial and tempo-
although a more daring goal may also be outlined. Indeed ral changes (Hunter, 2005; Leese et al., 2001).
there are many examples even in reproductive biology that (iii) In contrast to somatic cells that are unable to prolif-
the natural way is not necessarily the optimal or the most erate without external trophic ligands, mammalian
efficient one. In cattle, the overall efficiency of in-vitro em- zygotes are intrinsically programmed to develop to
bryo production is about 100-fold higher than the in-vivo the hatched blastocyst stage without any external
process if the developmental rate is measured from the an- stimuli. However, in vivo these embryos are exposed
tral follicle stage or from the spermatozoon to the blasto- and certainly respond to a number of interrelated
cyst. The latter difference can be even higher in humans endocrine, paracrine and autocrine factors. The com-
when intracytoplasmic sperm injection is applied. plex regulatory mechanism provided by these ligands
This review argues that mammalian embryo culture seems to be supportive for healthy development
in vitro should not be regarded as an imperfect copy of (O’Neill, 2008).
the in-vivo procedures, but an artificial process with its (iv) The oviductal and uterine fluid is not just the product
own frames, limitations and possibilities. Acknowledging of the epithelial cells. It contains factors derived from
the current limitations must not restrict the search for fu- the maternal body, reflects its actual status and may
ture perspectives. By using in-vitro systems, endless choices have profound consequences on the future of the
are offered – even within, but especially behind, the tradi- fetus and offspring (Leese et al., 2008a). The role of
tional ‘medium, gas, temperature, oil’ frames – to estab- oviductal and uterine epithelial cells includes a pas-
lish conditions and involve steps that fully exploit all sive filtration or active transport of these factors as
resources of mammalian embryo development, including well; this role cannot be reconstructed by a simple
those not used by the natural process, or to defend embryos monolayer of a co-culture system.
from a potentially or definitely harmful maternal effect. (v) The oviductal–uterine environment is not necessarily
The aim of this work is to provide a focused overview supportive to the embryo development. Depending on
about the state-of-the-art of mammalian, predominantly the status of the reproductive tract or the whole
human, embryo culture. Instead of detailed discussion of maternal body, it may also be harmful and may cause
all published achievements, approaches and theories, this serious damage with long-term negative effects on
review attempts to outline major trends, confront con- the development of the fetus or offspring (Kwong
tradicting opinions and call attention to some promising et al., 2000; Leese, 2002; Leese et al., 2008b; Oliver
new perspectives. et al., 2007).
Improving embryo culture 455

(vi) The establishment and maintenance of the microenvi- Unfortunately, a simple list such as the first one in the
ronment plays a crucial role in embryo development previous paragraph can summarize everything when a broad
both in vivo and in vitro (Hunter et al., 2003; 2005; consensus exists. Considering the invested energy and the
O’Neill, 2008). Autocrine factors seem to be decisive responsibility related to the procedure, the list is disap-
constituents of this microenvironment. With the cur- pointingly short and unspecific. Regarding composition and
rent methods, tools and dishes (established originally application of media, leading scientists of the field seem
for somatic cells), this microenvironment cannot be to disagree in all details. Not a single component is used
properly maintained in vitro. in exactly the same concentration in all commonly applied
human embryo culture media. There are marked differences
The other group of problems is related to the autocratic in concentration of the simplest elements, such as potas-
approach of dictating to embryos what they should prefer, sium chloride and magnesium sulphate (Gardner and Lane,
either by interpreting wrongly the in-vivo situation, or by 2004). The simple media that have been established and
applying its features mechanically to the in-vitro culture called analogues of oviduct fluid only marginally mimic
Examples for this mentality are listed in the following the natural oviduct environments and differ from each other
points: in almost all parameters (Biggers, 2001). Even the optimal
osmolality for development of human embryos in culture
(i) The existence of some commonly cited oviductal fac- has not been determined yet (Gardner, 2008). Moreover, al-
tors or phenomena has not been confirmed in vivo in most all media require supplementation with chemically
pregnant mammals, especially not in humans. undefined or partially defined factors as albumin or serum.
(ii) Several parameters measured as physiological constit- Components of media were described in detail in reviews
uents of the oviductal fluid in vivo may be detrimental of Bavister (1995), Biggers (2001), Biggers and Summers
in vitro (for example potassium level; Biggers, 2001), (2008), Gardner and Lane (2004), Gardner (2008), Leese
whilst others, not present in the oviduct, may not (2003), Mortimer (2001) and Yovich and Grudzinskas
have the widely supposed harmful effect in vitro. (1990). The summary presented here will focus only on
(iii) Some factors around and even inside the embryos the main tendencies and disputable details.
(e.g. hyaluronic acids in media, or intracytoplasmic The first successes of embryo culture were achieved in
lipid droplets in pigs) may not be required in a given the mouse with a simple Krebs–Ringer bicarbonate medium
in-vitro system. (Tyrode’s medium) supplemented with lactate (McLaren
(iv) Any intervention based on in-vivo analogues and per- and Biggers, 1958; Whitten, 1957). For initial human appli-
formed with the best intentions may have adverse cation, complex media (e.g. Earle’s balanced salt solution
effects that counterbalance, even reverse the or Ham’s F-10) were used; however, supplementation with
benefits. maternal serum was indispensable (Edwards, 1981).
(v) The selection of the mouse as the animal model is a During the subsequent decades, two major approaches
convenient solution for the scientist, but may be a were applied to develop media purposefully for human em-
suboptimal choice and culprit for erroneous practices bryos, both of them using simple media as starting points.
where human embryos are concerned. The first approach was the ‘empirical optimization’ of com-
(vi) For almost all embryologists, the elimination of any ponents by bioassays – also known as ‘let the embryos
kind of stress is the unquestionable principle of the choose’ principle – established by Biggers (KSOM-Global
optimal embryo culture system. However, a small family; (Biggers, 2001). The other way was the ‘back to nat-
but increasing number of data show that a precisely ure’ principle, i.e. to make media with composition similar
timed and appropriately, provided shock may induce to the oviductal fluid, outlined and baptized by Leese, 1998,
metabolic processes that eventually result in but applied previously to develop solutions including the
improved embryo quality and developmental HTF-Sage and STF-Cook family of Quinn and Mortimer,
competence. respectively (Mortimer, 1986; Quinn, 1995; 2000; Quinn
et al., 1985). However, none of these strategies was suc-
In the following sections, an attempt will be made to jus- cessful enough to be applied purely and exclusively for
tify the above statements and to provide several practical development of media.
suggestions for future improvements The ‘empirical optimization’ approach, even with com-
puter modelling, would require astronomic numbers of
experiments without acknowledging some principles based
Chemical components on data obtained from in-vivo measurements. Accordingly,
some compromises in the interpretation of the mathemati-
Media for human embryo culture should contain the follow- cal models are required and many of the variables – includ-
ing components: pure water, common salts of four metals ing amino acid concentrations – need a kind of bulk
plus sodium bicarbonate as buffer, sodium salt of EDTA or handling (Biggers, 2001; Summers and Biggers, 2003).
other chelator, pyruvate and lactate, amino acids, macro- The major problem of the ‘back to nature’ principle (as
molecules and antibiotics. These principles have been also acknowledged by Leese et al., 2008a,b) is the extre-
established and widely accepted by the final decade of mely low amount of the fluid in the oviduct and uterus
the 20th century and contributed substantially to the dra- and the technical and ethical problems related to its collec-
matic increase in efficiency of human assisted reproduction tion and measurement. So far, investigations have been per-
all over the world around the turn of the millennium (Quinn, formed in vivo (by micropuncture, chronic or acute in-situ
2004). canulation) or in vitro (by vascular and luminar perfusion
456 G Vajta et al.

or excision and rapid sampling; Leese et al., 2008a,b). How- detectable changes even before hatching, but most of them
ever, many of these methods cannot be applied to humans remain hidden from stereomicroscopic investigations.
and virtually none of them can be used to collect samples Considering that developmental anomalies occur in the
during the critical period of early human pregnancy; there- majority of the handful of species studied in detail so far,
fore, all conclusions are based on analogues. in-vitro culture of human embryos could be an extremely
A third approach, to use nutrient consumption data of risky and morally unacceptable challenge. However, the
the embryo, was also listed in the review of Leese (2003). everyday practice does not support this conclusion. There
Retrospectively, this latter approach was at least partially is only a very slight, if any, increase in the proportion of
used by all researchers who achieved successes in this field perinatal complications and developmental abnormalities
and was the basic element of the work of Gardner and Lane compared with natural reproduction, and none of these
(2004) that led to the introduction of the G-Vitrolife family pathologies can be definitely attributed to the in-vitro cul-
of media. ture (Chang et al., 2005; Khosla et al., 2001b; Paoloni-Gia-
The final result of these efforts could be a colourful pal- cobino, 2007).
ette of possibilities, with a good chance for the optimal Various explanations have been proposed attempting to
solution to evolve almost spontaneously, by a kind of natu- explain these differences between species, especially the
ral selection. However, there are two serious problems that privileged position of humans. The most concise summary
hamper this advancement. The first can be attributed to the is that there is no clear idea of the reasons. Retrospectively,
status of the human embryo in vitro, with various moral and it may be interpreted as a pure chance that humans belong
legal aspects drastically restricting the possibility of com- to the lucky minority. However, this generous gift of nature
parative studies. can only be accepted while keeping in mind that there are
The second is related to the aspect of commercializa- limits of such tolerance. These limits may crossed at any-
tion. More than 80% of major producers fail to supply infor- time, but the consequences may only be realized retrospec-
mation about the exact composition of their media; some tively some years or even decades later.
do not even provide a list of components. As stated by Big-
gers (2000) this approach may reduce presentations and pa-
pers to uncritical advertisement. Comparisons may only Physical constituents
happen between brand names and code numbers, the real
research work becomes the privilege of the inner circle of Media for human embryo culture are usually placed into
a company making a worldwide unified effort impossible. polystyrene dishes, in 10–80 ll drops covered by oil, then
placed in humid dark areas into a mixture of oxygen, carbon
dioxide and nitrogen with a consistent 37C temperature.
Sword of Damocles This system can be called anything but imaginative. Simplic-
ity, practicality and repeatability are its advantages, but
There are alerting new data underlining the importance of these features are to the benefit of embryologists and not
such worldwide efforts. The first week of mammalian em- the embryos.
bryo development consists of crucial events including the Most common dishes applied for human embryo culture
first cleavage, activation of the maternal genome, compac- are 30- or 60-mm diameter Petri dishes, less frequently
tion of morulae and differentiation with blastocyst develop- four- or five-well dishes. None of them has been developed
ment (Lonergan et al., 2006). All these steps are the result for embryological purposes. Special surface coatings may be
of precisely programmed and orchestrated events. Adapta- applied in some of these dishes, without proven benefit for
tion to an inappropriate environment may cause changes mammalian embryo development. Very recently, dedicated
in epigenetics, transcription, metabolism and cell alloca- dishes for mammalian embryo handling and culture have
tion. Components of media can produce serious alterations been produced (Rieger et al., 2007), but conclusive evi-
in the gene expression pattern of preimplantation mamma- dence regarding practicality, cost-efficiency and especially
lian embryos (Duranthon et al., 2008; Lonergan et al. 2006; the improvement in pre- and post-implantation develop-
Thompson et al., 2007; Wrenzycki et al. 2005) with poten- ment is still lacking.
tial long-term consequences. Oil has the bad reputation of being the least stable, most
In sheep, serum supplementation may cause up to three- sensitive element of the physical environment, which may
or four-fold increases in birthweight, higher incidence of become detrimental to embryos at anytime (Gardner and
abortions, neonatal deaths and post-natal malformations. Lane, 2004; Otsuki et al., 2007, 2009). Unfortunately, its
Similar phenomena were also described in less drastic form application in the current microdrop culture systems is
in cattle (Thompson et al., 2007), whilst in the mouse, de- indispensable both to physically separate drops from each
creased fetal weight, increased post-natal organ weight other and to prevent rapid evaporation, as well as changes
and exencephalopathy have been reported (Fernandez- of pH and temperature. Oils may be silicon- or carbon- (par-
Gonzalez et al., 2004; Khosla et al., 2001a,b; Lane and affin, mineral) based. They are usually supplied in sterile
Gardner, 1992; Lawrence and Moley, 2008; Zander et al., form and can be used without any treatment, although some
2006). In-vitro embryo culture for mouse has also caused laboratories prefer sterile filtration and/or washing and
behavioural problems (Ecker et al., 2004). Mechanisms in- equilibration with media.
volved are most probably related to epigenetic alterations Light sensitivity of embryos is one of the most disputed
involving imprinted genes, with placental abnormalities and least measured parameters (Oh et al., 2007). The toler-
and aberrant causal pathways as consequences (Thompson, ance may differ between species and developmental
2007). Some of the effects may cause morphologically phases. Mild exposure with a yellowish (Wolfram bulbs)
Improving embryo culture 457

low-intensity light from the ceiling or the microscope for a tains the minimum energy supply for one embryo over a 6-
limited period does not seem to have a detrimental effect. day in-vitro-culture period. This volume is more than the
However, exposure should be restricted to the minimum re- 10 ll medium suggested by Carolan et al. (1996) for individ-
quired level. ual bovine embryos. Later, Gardner and Lane (2004) re-
A general principle for the physical environment is to keep garded 50 ll drops for four human or domestic animal
it free from any uncontrolled insults. Possibilities include a embryos the minimum requirement, but with medium re-
broadly diverse scale from continuous mechanical vibrations newal every 48 h. Similar volume/embryo ratios are applied
to toxic fumes including highly volatile organic compounds. now worldwide in human IVF laboratories, although these
Authors fully agree with the opinion of Thouas (EmbryoMail, circumstances are far from supportive of communal effects.
28 August 2008), that the situation ‘... in a typical working Even the newly introduced commercially available dishes
clinical IVF lab with follicular fluid, blood and aerosols float- with both individual identification and communal culture
ing around all the time from multiple patients, with teams of possibility (Rieger et al., 2007) seem to contain too much
energetic embryologists, doctors, theatre staff and the pa- medium to benefit from the latter arrangement.
tients themselves moving around regularly’ is less than Curiously, sporadic empirical evidence indicates that
favourable to ensure the required calm consistency. Paradox- these commonly acknowledged volume/embryo rate limits
ically, a dull country laboratory in an old separate small build- are far from the real values. According to Ali (personal com-
ing may provide a much better environment, than a famous munication) continuous ultra-microdrop culture, i.e. 1.5–2 ll
university laboratory in the middle of a luxuriously equipped drops for culture of three to nine human embryos together
skyscraper and surrounded by cutting-edge research, educa- for 2–3 days. Both in-vitro and in-vivo developmental rates
tional status and medical facilities. were found significantly increased in the continuous ultra-
microdrop system compared with the control. Murine and
bovine embryos were also successfully cultured in closed
Questions to answer
systems containing 0.5–1 ll medium per embryo without
refreshment during the whole culture period (Roh et al.,
Only the most important questions are listed here, where 2008; Thouas et al., 2003; Vajta et al., 2001; see also
markedly different opinions between leading scientists ex- below).
ist, and where laboratories have to make their own Based on these results, a critical reconsideration of the
decisions. traditional opinion regarding the volume/embryo require-
ments is suggested. Modification of the culture system
Communal or individual culture? may eventually allow exploitation of the resources of the
group effect or may create a physical environment where
Microdrop culture offers an easy way to follow the develop- a similar effect for single embryos is also manifested.
ment of each embryo individually. The ultimate benefits of
the system are, however, questionable both for the embryos
and the embryologists. Most embryologists determine the Are co-cultures really needed?
fate of embryos (developing from normally fertilized 2PN
zygotes) at the last evaluation, just before transfer or cryo- Co-culture is becoming increasingly popular in human
preservation, regardless of the quality at the previous embryology (Desai et al., 2008; Mercader et al., 2006;
checkpoint. Therefore, individual identification is rather Parikh et al., 2006; Dominguez et al., 2008). A recent
important for administrative reasons or academic interests. meta-analysis of 17 prospective randomized trials has dem-
For the embryos, in most mammalian species they seem to onstrated that co-cultures increase blastomere numbers
dislike solitude. The ‘group’ or ‘communal’ effect has been and implantation, clinical and ongoing pregnancy rates
confirmed by numerous studies in mice, cattle and felids; (Kattal et al., 2008).
both in polytocous and monotocous species (summarized The fact that monolayers from various sources (e.g. pri-
by O’Neill, 2008; Vajta et al., 2000). In a routine bovine mary hepatocytes or even cell lines) can completely replace
in-vitro culture system, up to 50 embryos can be incubated oviduct and uterine cells for co-culture purposes indicates
together in 400 ll medium covered with 400 ll oil, in a well that the factors – if really present and contributing in the
of a four-well dish, grouping embryos close to each other in improvement – are not reproductive tract-specific. One
the middle of the dish. The consistently achievable 50% rate explanation for the beneficial effect is the decrease of the
of blastocysts per collected immature oocytes – compared oxygen concentration in the medium or the elimination of
with the 34–39% in individual drop culture – prove the va- other toxic factors (reviewed by Bavister, 1992; 1995; Don-
lue of this system and resolve concerns regarding the sup- nay et al., 1997). For the former, a decrease of atmospheric
posed negative effects of dead and dying embyos oxygen concentration from 20% to 5% eliminated the need
(Hagemann et al., 1998; Holm et al., 1999; Vajta et al., for co-cultures in cattle (Nagao et al., 1994; Trounson
2000). Gopichandran and Leese (2006) have even deter- et al., 1994; Voelkel and Hu, 1992). For neutralization of
mined the optimal distance between bovine embryos: other toxic factors, it may also play a limited role, as simpli-
165 lm, i.e. close to the commonly achieved average dis- fication of media by excluding unnecessary, potentially
tance when embryos are swirled together. toxic chemicals has definitely contributed to the tendency
Although the group effect can be increased by decreasing to abandon co-cultures in domestic animals (Thompson
the volume of the medium, current human culture systems et al., 2007).
do not exploit this possibility. According to Gardner et al. Accordingly, co-cultures seem to be beneficial when the
(1993), 20 ll of synthetic oviduct fluid (SOF) medium con- basic embryo culture is handicapped including the lack of
458 G Vajta et al.

communal effect. Unfortunately, a high price has to be paid the physical characteristics are only one, and maybe not the
for the benefits. Co-cultures have serious disadvantages, most important, feature of albumin. It may support embryo
including extra work and technical difficulties with standard development in many different ways, e.g. by modifying oxi-
establishment and handling, the risk of contamination, the dation of pyruvate (Eckert et al., 1999; Lee et al., 1998).
possible involvement of animal or human serum in phases Albumin also acts as a carrier for vitamins, hormones, bioac-
of isolation and culture; and the lack of appropriate control tive lipids and autocrine ligands (O’Neill, 2008). Addition-
over factors involved. Moreover, just like serum, co-cul- ally, albumin may neutralize some toxins occurring in the
tures were found to cause developmental abnormalities in culture media (Gardner, 2008). Enrichment of media with
cattle (Khosla et al., 2001b; Van Wagtendonk-de Leeuw more complex protein supplementation may result in fur-
et al., 1998). In the future, less complicated and more reli- ther improvement in blastocyst and implantation rates
able techniques are expected to replace communal effect (Meintjes et al., 2009a).
and to overcome problems for which co-culture seems to Albumin is the most common protein of the female gen-
be the best solution today. ital tract fluids. This fact is often cited when compared with
serum. On the other hand, the argument that serum is not
Oxygen concentration: 21% or 5%? physiological because embryos do not meet serum during
preimplantation development is not really convincing:
embryologists have welcomed many substances that help
In domestic animals, considerable confusion was created in
embryos to develop including EDTA and would welcome
the early 1990s by the identical observation of three inde-
PVA if its supportive effect would be comparable to serum
pendent research groups (Nagao et al., 1994; Trounson
or albumin, although neither EDTA nor PVA are natural con-
et al., 1994; Voelkel and Hu, 1992) that co-cultures of em-
stituents of oviductal or uterine fluid.
bryos with somatic cells require atmospheric oxygen con-
In the early decades of embryo culture, serum was a
centrations, while embryos without somatic cell support
common constituent of media. Its presence was especially
develop better in decreased oxygen. Various explanations
required for co-cultures, as somatic cells need serum sup-
of this phenomenon may exist, none of them was fully pro-
plementation for healthy growth. However, since the late
ven, and with the introduction of simple media and omission
1990s, serum has become the ‘pathological fluid’, a culprit
of co-cultures the whole problem has lost its significance.
that embryos should never meet during culture. Even short-
All successful research groups in cattle, pig, sheep and goat
term application was declared unwelcome.
embryology use simple media with supplementation and 5%
There are serious reasons to support this opinion but the
or 6% oxygen concentration, in complete accordance with
baby should not be thrown with the bath water, even if the
the in-vivo observations. Oxygen concentrations in oviduct
analogy is rather frivolous in this context. As mentioned
and uterus of all investigated species are considerably lower
above, in sheep (and to a lesser extent in cattle) presence
than the atmospheric oxygen concentrations (1.5–6% versus
of serum was found to cause morphological alterations of
21%, respectively). Unless convincing strong evidence oc-
preimplantation embryos and serious fetal, neonatal and
curs, the low oxygen concentration should be a principle
post-natal abnormalities, summarized as large offspring
for all mammalian embryo culture systems including humans
syndrome. One of the most cited studies also involved appli-
(Meintjes et al., 2009b). This is one of the few questions
cation of 20% human serum in sheep (Thompson et al.,
were a definite answer is available and a worldwide consen-
1995). However, Lawrence and Moley has stated recently
sus is being formed right now.
that ‘because of the change in media involved several vari-
Further decrease of the oxygen concentration in vitro
ables, it was not possible to definitely prove that human ser-
may have negative consequences. Oxygen at 2%, although
um was responsible for the large offspring syndrome of the
leading to increased blastocyst rates, may cause develop-
offspring’ (Lawrence and Moley, 2008). Additionally, large
mental abnormalities in ruminants (Thompson and Peter-
offspring syndrome occurs in sheep after in-vitro culture
son, 2000). On the other hand, there is no evidence that
without serum, in the presence of PVA or bovine serum albu-
embryos need a continuous gas supply. A gas mixture vol-
min as well (Rooke et al., 2006; Sinclair, 2008). Although
ume of 50 ml in a closed system generously covers the
Khosla et al. (2001b) have found an increase in developmen-
requirements of 200 bovine embryos for 1 week, from the
tal abnormalities in mice after culture in 20% serum, they
zygote to the blastocyst stage (Vajta et al., 1997).
also state that only few studies have dealt with this phe-
nomenon, which is surprising compared with the extensive
Supplementation with macromolecules use of the mouse as the animal model for humans. A thor-
ough search of the available literature proves that most di-
Over decades, reports in both domestic animal and human rect evidence has been obtained from sheep (which is most
fields were repeatedly published about completely defined probably predisposed to large offspring syndrome) or from a
culture systems that approach or even reach the overall very limited number of studies performed in other species.
efficiency of those supplemented by proteins. Moreover, reviews often talk about general effects of serum
The most frequently used chemically defined macromol- without specifying the species of embryos, the origin of ser-
ecule supplement is polyvinyl-alcohol (PVA), as it provides um or the applied concentration.
surfactant activity similar to albumin (Thompson, 2000) The latter seems to be very important. All cited studies
and prevents gametes attaching to each other or any sur- are based on very high (15–20%) concentrations of serum,
face they contact (dish, pipette, tubes, etc.) during in-vitro disregarding other possibilities. However, the observations
manipulation. Another common feature is the maintenance of O’Neill (1997) that nanograms of albumin are more
of the colloidal osmotic pressure (Gardner, 2008). However, efficient to support embryo development than the usual
Improving embryo culture 459

mg/ml formulae may also be valid for serum, at least to (Yoshioka et al., 2002), respectively. These media with ser-
some extent. Supplementation of modified synthetic um or albumin supplementation are capable of supporting
oviduct fluid (SOFaaci) medium with 5% cattle serum allows 60–90% blastocyst development from parthenogenetically
blastocyst production from 50% of collected immature activated in-vitro-matured oocytes. The common special
cattle oocytes after in-vitro maturation and fertilization. features of these media include: (i) they are single media
These blastocysts are morphologically similar to those from zygote to blastocyst stage; (ii) none of them contains
developing in vivo and are without the pathological features glucose or EDTA; and (iii) the potassium content is relatively
(accumulation of lipid droplets, mitochondrial degenera- high. As suggested by Bavister (2002) more intensive dia-
tion) observed after 15–20% supplementation. The cryotol- logue between human and domestic animal embryologists
erance of embryos cultured in 5% cattle serum was may eventually lead to useful changes in the composition
comparable with their in-vivo counterpart (Vajta et al., of human media.
1998). In pilot studies, no increase of large offspring after Data obtained from domestic animals may also help to
transfer of these embryos was observed (G Vajta et al., establish a more balanced opinion regarding the need of
unpublished data; Lewis and Callesen, unpublished data). medium change during preimplantation embryo develop-
According to Thompson et al. (2007), patient serum was ment in vitro, especially in light of the fact that most hu-
widely used in the early days of human IVF with no apparent man-research scientists completely disregard that neither
dire consequences. SOF nor PZM requires medium renewal during the whole cul-
Application of serum for several embryological proce- ture period (see below).
dures including cryopreservation has been found beneficial
and difficult to replace with additives of known composi-
tion. Unknown factors present in serum (e.g. endocrine li- Renew medium or not?
gands) may also have a supportive effect for embryos.
In some species (e.g. cattle, as discussed above), low In their extensive review listing many arguments for sequen-
concentration of serum may be a better choice than albu- tial versus single media, Biggers and Summers (2008) also
min while, in pig, addition of bovine serum albumin seems mention the non-renewal of media as a possibility with po-
to be superior to serum (G Vajta, unpublished data). In tential benefits. However, in spite to their earlier good re-
the widely used human embryo culture systems, supplemen- sults in the mouse (Biggers et al., 2005), they seem to
tation with human serum albumin seems to be appropriate. avoid dealing with the non-renewal in detail. The possibility
However, further investigations are required including crit- has been listed in one of their tables, describing many posi-
ical overview of available data to find the optimal macro- tive features of this approach: (i) the embryos are left
molecule supplementation. Eventually, serum or some of undisturbed; (ii) accumulated endogenous growth factors
its constituents may become useful elements of many tech- are left in place; (iii) the relative environmental stress is
niques in human embryology, and after thorough clarifica- ‘low’ (iv) labour intensity is lower; (v) cost is lower; and
tion of safety issues, the future application of these (vi) less quality control is required. Two possible disadvan-
constituents as substitutes in embryo culture media cannot tages are also mentioned: essential nutrients are not re-
be excluded, either. placed and toxins may accumulate.
The former disadvantage should not be a major problem.
If single media are accepted as appropriate for the full in-vi-
Sequential or single medium? tro period, the supply with nutrients is only the question of
volume/embryo ratio. As mentioned earlier, according to
This is probably the most exciting question in human embryo data of Roh et al. (2008), Thouas et al. (2003), Vajta
culture today. Papers supporting either sequential or single et al. (2001), murine and bovine embryos may achieve high
media have been published repeatedly (Gardner and Lane, blastocyst rates in medium volumes that are with an order
1997; 2003; Gardner, 2008; Gardner and Lane 2007; Lane of magnitude lower than those routinely applied in humans
and Gardner, 2007; Pool, 2002; 2005; versus Biggers, 2001; (0.5 ll and 1 ll per embryo during the whole culture period,
Summers and Biggers, 2003; Biggers et al., 2005; Biggers for mice and cattle, respectively).
and Summers, 2008; Sepúlveda et al. , 2008, see also direct For the possible accumulation of toxic substances, the
arguments: Gardner and Lane, 2006 versus Biggers et al., same publications may be referred to. Theoretically, mate-
2006). While most human IVF laboratories use some kind rials with potential negative effect may originate from dif-
of sequential media today, there is an increasing group of ferent sources, including dead embryos in communal
scientists preferring the latest versions of single media. This culture, ageing of the medium, metabolism of healthy em-
review refrains from making any general judgement. The bryos and diffusion of toxic substances from the oil, atmo-
discussion of the problem will be restricted to listing the sphere or plastic dish. Again, theoretically, medium
published pros and cons (Table 1), to adding some new or exchange may keep accumulation of these toxins under con-
usually disregarded points and to adding personal opinion trol. However, the common laboratory practice does not
where it seems to be relevant. support the idea. There are no data or signs that dying or
At present, the overwhelming majority of data used for dead embryos hamper the development of their neighbours.
development of commercially available human media are In an appropriate embryo culture system, the concentration
derived from experiments performed on murine embryos. of toxic substances derived from the environment should be
On the other hand, successful embryo culture media have basically low and their control should not require medium
been developed in cattle and pig including SOFaaci (Holm change. Regarding the metabolism of normal embryos and
et al., 1999) and the porcine zygote medium (PZM) family the spontaneous degradation of medium components, the
460 G Vajta et al.

Table 1 Arguments for sequential media versus a single medium.

Sequential media Single medium

Evidence
In-vivo data obtained from humans show different There are no direct data obtained from pregnant women
concentrations of pyruvate, lactate and glucose in the (Biggers, 2001)
oviduct and the uterus, changing also during the menstrual The metabolism of the embryo is not strictly determined by
cycle (Gardner et al., 1996) the environment. In vitro, embryos may adapt to constant
environment with their changing metabolism as long as the
constituents fall between tolerable ranges (Biggers and
Summers, 2008)

Carbohydrate metabolism
In-vitro cleavage-stage mouse and human embryos use Pyruvate in 2 mmol/l concentration seems to be sufficient to
pyruvate as primary energy source, while glucose after the support embryo development in vitro (Biggers, 2001). There
8-cell stage (Leese and Barton, 1984; Leese et al., 1993). are no data about harmful effects of this concentration of
Pyruvate values in the oviduct are higher than in the uterus pyruvate to further embryo development
(Gardner and Lane, 1996) Glucose is present in a considerable concentration at any
Glucose may have an inhibitory effect on development of phase of the cycle in human oviduct and uterus (0.5 mmol/l
cleavage-stage mammalian embryos including humans and 3.15 mmol/l, respectively (Gardner et al., 1996).
(Schini and Bavister, 1988; Conaghan et al., 1993). Complete omission of glucose is unlikely to benefit the
Accordingly, in the first interval, glucose should be reduced embryo as glucose plays an important role in ribose synthesis,
to 0.5 mmol/l or omitted completely (Pool, 2002; 2005) which essential in early development as well (Thompson,
2000). Media with up to 4.7 mmol/l glucose concentration for
the entire culture period may support mammalian embryo
development including humans (Summers and Biggers, 2003)

EDTA
Ethylenediamine tetraacetic acid (EDTA) supports early The harmful effect of EDTA in 0.01 mmol/l concentration was
embryo development to overcome the 2-cell block in mouse demonstrated only in cell-free extracts. Direct evidence
(Abramczuk et al., 1977) shows that 0.01 mmol/l EDTA during the whole culture period
The generally acknowledged efficient concentration of does not impair embryo development in mouse (Biggers et al.,
EDTA (0.01 mmol/l) may compromise glycolytic activity in 2005; 2006)
the inner cell mass; therefore it is potentially harmful for
further embryo development (Lane and Gardner, 2001;
Gardner and Lane, 2006)

Glutamine metabolism
Ammonium accumulation in the medium, as the result Occurrence of exencelopathy was never confirmed in the
predominantly of glutamine deamination, has been shown same system or in other animal models with similar glutamine
to induce exencephaly in mice (Lane and Gardner, 1994a,b) concentrations (Biggers and Summers (2008))
Addition of exogenous ammonium impairs embryo Exogenous addition of ammonium may not follow the
development and induces exencephaly (Lane and Gardner, kinetics of glutamine breakdown (Biggers and Summers,
2003) 2008). Ammonium may be more harmful for early-stage
When high concentration of ammonium was measured in the embryos, while at the end of culture period when ammonium
culture medium, human embryos had impaired accumulation may occur, embryos are relatively insensitive
development (Virant-Klun et al., 2006) (Zander et al., 2006)
Glutamine can be replaced by stable dipeptides of
glutamine which do not give rise to ammonium (Biggers et al.,
2004)
The volume of culture medium was unusually high (0.5 ml
for small groups or single embryos) and was changed every
day. It is not clear whether increased ammonium was the
cause or the consequence of compromised embryo quality.
The system does not seem to provide a strong argument for
sequential media (G Vajta, unpublished data)
Improving embryo culture 461

Table 1 (continued)

Sequential media Single medium

Amino acids
Non-essential amino acids stimulate cleavage between the Human embryos make no distinction between essential and
zygote and 8-cell stage and consequently facilitate non-essential amino acids (Leese, 2003)
blastocyst formation and hatching (Gardner and Lane, It is prudent to add a full mixture of 20 amino acids,
1993; Lane and Gardner, 1994) according to the composition of the tubal fluid (Leese, 1998;
Essential amino acids that are in low concentration in Tay et al., 1997)
oviduct appear to reduce cell numbers of blastocysts, by
inhibition during the first 4-cell cycle (Gardner and Lane,
1993; Steeves and Gardner, 1999)

only material that was supposed to have detrimental effect their changing metabolism and supposedly changing
is ammonium: by replacing glutamine with stable dipeptides requirements. Unfortunately no significant increase in the
(Biggers et al., 2004), this danger can be safely eliminated. embryo developmental rates was obtained. Subsequent ver-
In conclusion, a single medium without renewal may be a sions of the system (summarized by Thompson, 2007) also
realistic alternative of the present culture systems. It may failed to demonstrate benefits over the traditional culture
offer many practical benefits for embryologists and it may methods and did not get wide acceptance for practical use.
also help embryos to build up and maintain their
microenvironment.
Microchannel microfluidic system
Alternative possibilities for embryo culture
Microfluidics is a multidisciplinary approach developed ini-
tially for inkjet printers in the 1980s but soon applied for var-
In sharp contrast to the enormous efforts invested to im- ious purposes in physics, chemistry, microtechnology and
prove culture media, surprisingly few attempts have been biotechnology, including the lab-on-a-chip for biotechnol-
made to establish the appropriate tool to hold embryos dur- ogy. The principle is that no turbulence occurs in small chan-
ing in-vitro development. One consequence of the adoption nels (from 100 nm to several hundred m), i.e. the flow of
of monolayer culture systems for embryo culture was the solutions remains laminar and the components mingle by dif-
acceptance of the two-dimensional approach, confirmed fusion only. This special characteristic helps to control vari-
also by the stereo- and inverted microscopes. Although this ous manipulations. With the use of computer-controlled
approach has simplified all manipulations, it provided a sub- pneumatic valves and micropumps, a rather complicated
optimal environment for globes that are completely sur- system can be designed, resembling an electric integrated
rounded in vivo by the microvilli of the oviductal circuit, and the microchannels may be made suitable for var-
epithelium. Until recently, attempts to exploit the potential ious manipulations. Macroscopically, the device usually con-
benefits of a three-dimensional embryo culture system were sists of a glass microscope slide base and a plastic (e.g.
sparse and none of the recently introduced alternative polydimethylsiloxane) layer with the channels and valves
methods has obtained wide acceptance. connected with outlets to mechanical or automatic pumps.
Microfluidics offer an excellent possibility to establish
Tubes and maintain optimal microenvironment for embryos. How-
ever, further potential applications include almost all steps
The simplest three-dimensional culture system was applied of human IVF, e.g. oocyte maturation, sperm selection,
first by Trounson and Conti (1982) and Bavister et al. cumulus or zona removal, in-vitro fertilization, embryo cul-
(personal communication) by placing embryos in common ture with or without medium change and zona thinning. Fur-
test tubes for the whole culture period. Although the system thermore, microfluidics are also capable for complicated
was practical and easy to use, the lack of the possibility to tasks including fluorescence activated cell sorting (reviewed
visually follow-up embryo development was a major disad- by Beebe et al., 2002; Smith and Takayama, 2007; Thomp-
vantage for human embryologists. Very recently, a similar son, 2007; Vajta et al., 2004). As integration of the manip-
system with 250 ll PCR microtubes, 10 ll volume and 20 ulations into a production line is also possible, there is still a
mouse embryos per tube and without oil overlay was pub- theoretical but quite realistic chance of making complex
lished by Roh et al. (2008) and the obtained blastocyst rates procedures completely automated, including the whole hu-
were higher than in the traditional drop culture system. man IVF procedure and even somatic cell nuclear transfer
Thompson (1996) have established a more sophisticated (Smith and Takayama, 2007; Vajta et al., 2004).
system with the definite purpose to improve efficiency. By Although the first commercially produced versions of
using a modification of an early idea of continuous perifu- microfluidics are already available, the widespread applica-
sion (Deter; 1977) a semi-automated perfusion culture sys- tion is hampered by simple technical problems (Thompson,
tem was created by culturing embryos in tubes. The 2007). Additionally, in the field of embryo culture, almost
system provided embryos the optimal culture medium at all published applications have exploited one of the most
all phases of preimplantation development according to fascinating intrinsic potentials of the microfluidic technique,
462 G Vajta et al.

the continuous or stepwise medium exchange. However, it to those achieved in communal culture (Vajta et al., 2000).
may not be the optimal approach, as proven by the success Later studies have shown a dramatic increase in developmen-
of the glass oviduct and Well of the Well systems. tal rates of parthenogenetically activated pig embryos and
increased speed of development of in-vivo-produced mouse
Glass oviduct system zygotes (Vajta et al., 2008). A very recent study has demon-
strated that gene expression patterns of bovine embryos
The glass oviduct (GO) system (Thouas et al., 2003) can be cultured in WOW show closer resemblance to in-vivo-derived
regarded as an extremely simplified microfluidic device embryos than those cultured on flat surfaces (Ghanem et al.,
that provides a completely static environment. It is based 2009). Similar microwell systems produced in agar gels have
on an open-ended 2 ll sterile capillary with 200 lm inner also been reported to be successful in various animal models
diameter. Loading of the embryos is performed manually (Peura and Vajta, 2003; Thompson, 2007).
by immersing one end of the capillary in a Petri dish con- The first human application has resulted in higher blasto-
taining the embryos in a standard microdrop system. While cyst rates when sibling embryos were cultured in WOW ver-
passing through the oil layer, a small oil column enters into sus traditional culture (56% versus 37%, respectively).
the glass tube as the result of the capillary effect, fol- Pregnancy and birth rates achieved with embryos cultured
lowed by the medium and one or two embryos, for mice in the WOW system are still preliminary but very promising
or cattle, respectively. Upon retraction of the capillary, (Vajta et al., 2008).
a small oil column enters again; therefore, the approxi- The mechanism explaining the successes of the GO and
mately 1 ll of medium inside the capillary is separated WOW systems still requires further clarification. The sys-
from the atmospheric environment at both ends by oil. tems’ common features are the small amount of medium
The capillary is inverted subsequently and cultured verti- surrounding the embryo and the static or semi-static
cally for the entire culture period undisturbed in a carbon arrangement. Both factors allow the establishment of a
dioxide incubator. Comparing the system with a standard stable microenvironment. In the GO system, the embryos
microdrop technique, similar blastocyst rates were sink to the oil–medium border and are closely surrounded
achieved in mice, but the cell numbers and hatching rates by 0.1–0.2 ll medium; the thin column in the capillary
were higher in the GO system. The efficiency of the tech- allows considerable concentration gradients for factors
nique was also confirmed in cattle, both for zona-intact selected or eliminated by the embryos. The volume with
and zona-free embryos cultured individually (Vajta et al., WOW is approximately 0.05 ll. Although the system is open
2001; G Vajta, unpublished data). upwards, it may serve as a nest for the embryos, to establish
and maintain the optimal microenvironment and to mini-
mize the need of constant adaptation. It is also worth con-
Well of the Well system sidering that the size and shape of the WOW may have
considerable effect on the outcome: the deeper and
An alternative solution to use the benefits of the three- narrower the WOW, the better embryo development can
dimensional arrangement is the Well of the Well (WOW) sys- be achieved (Feltrin et al., 2006).
tem (Vajta et al. 2000). It consists of a small microwell of Various factors were supposed to contribute to the ben-
conical shape, 200–300 lm diameter and depth, produced eficial effect of an appropriate microenvironment (re-
in a well of a four-well dish or in a Petri dish. viewed by; Gandolfi; Thompson, 2000; Vajta et al., 2000;
The system was originally established for in-vitro culture Richter, 2008). An excellent detailed analysis of both com-
of zona-free cloned embryos to keep the blastomeres to- munal culture and microclimate has been published by
gether before compaction. It was based on the darning nee- O’Neill (2008). He concludes that the communal effect
dle hole system used previously for aggregation chimeras may create both deleterious and beneficial effects, with
(Wood et al., 1993). However, the requirements of a short a net beneficial outcome, and that much of the beneficial
aggregation and a long embryo culture are different. The effect is accounted for by the action of autocrine trophic
latter needs larger and deeper wells to avoid floating out ligands in defined culture media. The effect can be in-
of embryos and smooth walls to permit visual evaluation duced by increasing the number of the embryos in a given
of their development. The possible repeated movements medium volume or by decreasing the volume that sur-
of the dish also require larger wells. These modifications rounds the embryo(s). The review, based on his previous
were achieved first by melting the plastic (Vajta et al., work, provides convincing evidence, and also a detailed
2000), later with a modified mechanical method (Booth analysis of the possible molecular mechanism of this auto-
et al., 2001; Vajta et al., 2005). With all these modifica- crine ligand effect, showing that it may have a critical role
tions, the first goal, i.e. culture of zona-free embryos from – together with paracrine and endocrine factors – in
the zygote to the blastocyst stage) was fully accomplished embryo development in vivo as well.
and the system has contributed significantly in the success The lack of a breakthrough with sophisticated perfusion
of the handmade cloning technique (reviewed by Lagutina systems, the successes achieved with simple static culture
et al., 2007; Vajta et al., 2005; Vajta et al., 2007). methods (GO, PCR tubes, WOW), the commonly acknowl-
Another application possibility of the WOW system is to edged communal effect and its molecular explanation are
use it for zona-enclosed embryos. Although it is an individual all strong indicators of the importance of microenviron-
culture system, it is suitable to replace the communal ef- ment. It cannot be further neglected in practical work.
fect. In cattle, culture of single embryos in WOW resulted When designing embryo culture systems, a priority should
in a considerable increase of blastocyst rates compared with be given to the maintenance of the milieu that embryos cre-
drop culture (from 32% to 60%), and the results were similar ate themselves.
Improving embryo culture 463

Table 2 Differences and similarities between embryos of some mammalian species.

Characteristic Mice Cattle and sheep Pigs Humans

Volume of the zygote, 5–8 times Similar to human Similar to human –

metabolic reservesa smaller than
human

Cytoplasmic lipid content Low High Extremely high Moderate

Development to blastocyst On day 4–5 On day 6–8 On day 5–6 On day 5–6

Embryo genome activationb 2-cell stage 8–16-cell stage 4–8-cell stage 4–8-cell stage

Amino acid metabolismc Different from Different from humans Similar to humans –
humans

Pyruvate/lactate versus glucosed Switches to No absolute need for No absolute need for No absolute need for
glucose at 48 h glucose before glucose before glucose before
hatching hatching hatching

Overall sensitivity in vitro Low High Extremely high High

Genome sequencing Completed Proceeding Almost completed Complete

Genome structuree Far from Unknown Close to humans –

humans

Demethylation and methylation Extensive Considerable (cattle), Moderate Probably moderate

during early embryo moderate (sheep)
developmentb

Time and location of embryo Exact match Exact match required Flexible Flexible
transfera required

Developmental anomalies after May occur Frequent and serious Very rare Very rare
in-vitro culture
a
Leese, 2003.
b
Duranthon et al., 2008.
c
Booth et al., 2005.
d
Bavister, 1995; Vajta et al., 2004
e
Wernersson et al., 2005.

Alternative animal model available only for a handful of these species, a compara-
tive analysis of relevant factors may demonstrate that
Many practical characteristics predispose the mouse to be- some of them may be more suitable for special purposes
come the animal model for almost anything in mammalian of embryology research. The main evaluation points are
biology. Accordingly, selection for early embryological re- summarized in Table 2.
search was evident and has led to fundamental discoveries. According to these data, pig should be considered as an
Quinn and Horstman (1998) have acknowledged its use as alternative animal model for human embryo culture. This
model animal for human embryology, justifying the prac- suggestion meets the overall tendency in medical research
tice of the previous decades. However, the fact that al- (Vodicka et al., 2005). Moreover, during the past 5 years,
most everything that is going on today in human embryo the efficiency of porcine embryo culture has improved expo-
culture is based on murine experiments may be an exag- nentially, mainly due to changes that have also increased
geration of the relevance and applicability of this species efficiency in humans, but without sequential culture and
(Betteridge and Rieger, 1993; Ménézo and Hérubel, without medium change. Today, blastocyst rates with par-
2002). Recent research has proven the existence of funda- thenogenetically activated in-vitro-matured oocytes may
mental differences in culture requirements between mam- reach 60–90% (Vajta et al., 2005). Finally, some common
malian species. Although appropriate embryo culture is features between mice and pigs (relatively short pregnancy
464 G Vajta et al.

compared with the size of the latter species, large litter, erably cooler than the surrounding tissues, and the differ-
relatively low costs and experience in breeding) and the ence is greater around ovulation and around mating
easy access to oocytes by using slaughterhouse-derived ova- (Hunter and Nichol, 1986; Hunter et al., 2000; Hunter
ries also support the feasibility of changing the model sys- et al., 2006; Leese et al., 2008b). The mechanism of this
tem, or at least involving pigs in tests when improvements strange cooling mechanism inside a warm body requires fur-
of human embryo culture are attempted. ther research, but the consequence of the lower tempera-
ture is most probably a decreased rate of embryo
Quietness versus stress metabolism, and possibly a better embryo quality.
For the practical work in human embryology, this obser-
In 2002, in a much cited paper, Leese launched the follow- vation may indicate that a radical change is needed in the
ing hypothesis: ‘. . . preimplantation embryo survival is best temperature of all incubators, workstations, heated
served by a relatively low level of metabolism; a situation stages, dishes, tube heaters etc. These instruments are
achieved by reducing the concentrations of nutrients in cul- now scrupulously adjusted to 37C, the routine calibration
ture media, and encouraging the use of endogenous re- and control is one of the most important quality assurance
sources’. According to the author (and supported by many activities in a human assisted reproduction laboratory. A
previous observations), the consequence of in-vitro embryo preliminary study compared blastocyst development in cat-
culture is increased metabolic activity and compromised tle at 37C with the conventional 39C and resulted in the
developmental competence, even if the final consequence same blastocyst rate but lower amino-acid turnover, a qui-
is manifested only in the fetal or post-natal phase. Qualita- eter state that may be interpreted as a more in-vivo-like
tive assessments including higher glycogen content, reac- status (Sturmey et al., unpublished; cited by Leese
tive oxygen species formation and glucose consumption et al., 2008b).
in vitro support the hypothesis. On the other hand, reduc- The ‘quiet embryo’ theory has, however, been chal-
tion of metabolism by specific agents including low atmo- lenged by a series of recent observations. Based on the
spheric oxygen and chemicals blocking oxidative experiments of Wemekamp-Kamphuis et al. (2002) in bacte-
metabolism improve development (Gardner, 2008). It has ria, gametes and early embryos were exposed to high hydro-
also been observed that human embryos with reduced ami- static pressure (HHP) to induce a sublethal stress. The level
no acid turnover have higher potential to develop to blasto- of pressure varied between species and developmental
cysts (Houghton et al., 2002). phases, but was extremely high (between 20 and 80 MPa)
Another observation also supports the ‘quiet embryo’ compared with the pressures these cells meet under physi-
hypothesis. Ovaries and Fallopian tubes seem to be consid- ological circumstances. After exposure for 1 or 2 h and a

Table 3 Achievements with sublethal stress to induce stress tolerance in gametes and embryos.

Sample Stress Consequence Reference

Mouse in-vivo- HHP60 MPa, 30 min Improved development after slow-rate Pribenszky et al., 2005a
derived freezing
blastocysts
Bovine in-vitro- HHP 80 MPa, 45 min Improved development after slow-rate Pribenszky et al., 2005b
produced freezing
blastocysts
Bovine in-vitro- HHP 60 MPa, 60 min Improved development after vitrification Pribenszky et al., 2007a
produced
blastocysts
Porcine ejaculated HHP 30 MPa, 90 min Increased post-thaw motility and integrity Pribenszky et al., 2005c
semen
Porcine ejaculated HHP 30 MPa, 90 min Increased litter number after AI Pribenszky et al., 2008a
semen
Porcine ejaculated HHP 30 MPa, 90 min Increased litter number after Kuo et al., (2007)
semen cryopreservation and AI
Bovine ejaculated HHP 30 MPa, 90 min Increased post-thaw motility and integrity Pribenszky et al., 2007b
semen
Porcine in-vitro- HHP 20 MPa, 60 min Increased development after vitrification Pribenszky et al., 2008b;
matured oocytes and PA Du et al., 2008a
Porcine in-vitro- HHP 20 MPa, 60 min Increased development after vitrification Du et al., 2008b
matured oocytes and SCNT cloned offspring born
Porcine in-vitro- 1% (v/w) extra NaCl added, Increased development after PA or SCNT Lin et al., 2009a
matured oocytes 60 min
Porcine in-vitro- Osmotic stress with sucrose Increased development after PA or SCNT Lin et al., 2009b
matured oocytes and trehalose, 60 min

AI = artificial insemination; HHP = high hydrostatic pressure; PA = parthenogenetic activation; SCNT = somatic cell nuclear transfer.
Improving embryo culture 465

recovery period, gametes and embryos presented an im- ture can be considerably expanded in the future. The basis of
proved performance (increased fertilizing ability, develop- this opinion is that there are natural limits of advancement
mental competence after activation, IVF or somatic cell and that, essentially, the laws of nature cannot be crossed.
nuclear transfer, increased cryotolerance, etc.). Later, sim- As demonstrated by the present review, the authors
ilar results were achieved by applying osmotic stress instead respectfully disagree with this opinion. Present embryo cul-
of HHP. The detailed discussion of these observations ex- ture systems miss the consensus and solid scientific evi-
ceeds the scope of this review; the published achievements dence in basic elements, accordingly open debates
are summarized in Table 3. According to the preliminary challenging the currently applied principles may eventually
investigations performed in cattle, mice and pigs, the treat- result in considerable improvements. On the other hand, as
ment had no long-term consequence on sex ratios, malfor- mentioned above, there are proven possibilities outside the
mations, bodyweights and other detectable characteristics present frameworks that may result in radical changes in
of offspring. both quantitative and qualitative efficiency. To refer the
Very recently, independent research has led to a similar relevant words of one of the most creative experimental
conclusion: elevated temperature applied for hours after embryologists, Steen Malte Willadsen: ‘The role of the sci-
either in-vitro fertilization, parthenogenetic activation or entist is to break the laws of nature, rather than to estab-
somatic cell nuclear transfer has dramatically increased lish, let alone accept them.’ (Silver, 2007).
the in-vitro developmental potential of porcine embryos
(Isom et al., 2009). The earlier observation of Leach et al.
(1993) regarding the supportive effect of short ethanol Acknowledgements
treatment to the development of mouse embryos in vitro
may also be related to the events observed after HHP, os- The authors thank the sharp and most relevant critical re-
motic stress or elevated temperature. marks of Professor Ronald HF Hunter regarding an earlier
The phenomenon that a sublethal stress induces a re- version of this manuscript. They hope that this work now
sponse with temporary increase of a general, aspecific resis- more or less meets his high standards.
tance to various further stresses has been observed in almost
all levels of life, from bacteria to multicellular organisms
including humans (general adaptation syndrome; Selye, References
1998). On the cellular level, the reaction incorporates sens-
ing, assessing and then counteracting stress-induced dam- Abramczuk, J., Solter, D., Koprowski, H., 1977. The beneficial
age, consequently increasing temporary tolerance of such effect of EDTA on development of mouse one-cell embryos in
damage (Kültz, 2005). Factors that are involved in the key chemically defined medium. Develop. Biol. 61, 378–383.
functions of stress response are conserved in all cells and Bavister, B.D., 1992. Co-culture for embryo development: is it
participate in cellular functions including protein, DNA and really necessary? Human Reprod. 7, 1339–1341.
chromatin stabilization and repair, cell cycle control, redox Bavister, B.D., 1995. Culture of preimplantation embryos: facts
regulation, energy metabolism, fatty acid/lipid metabolism and artifact. Human Reprod. Update 1, 91–148.
Bavister, B.D., 2002. How animal embryo research led to the first
and elimination of damaged proteins (Kültz, 2003). Available
documented human IVF. Reprod. BioMed. Online 4 (Suppl. 1),
data are still insufficient to give a detailed explanation of
24–29.
which factors are involved in the stress response of gametes Beebe, D.J., Wheeler, M., Zeringue, H., et al., 2002. Microfluidic
and embryos. Nevertheless, it is rather surprising that, in technology for assisted reproduction. Theriogenology 57, 125–
spite of the relative silence in transcription and translation 135.
in gametes and early embryos, they can react so rapidly Betteridge, K.J., Rieger, D., 1993. Embryo transfer and related
and efficiently to stress. techniques in domestic animals, and their implications for
In summary, controversial observations have created a human medicine. Human Reprod. 8, 147–167.
new question: to stress or not to stress? The right answer Biggers, J.D., 2000. Ethical issues and the commercialization of
might be that in general, stress, especially in uncontrolled embryo culture media. Reprod. BioMed. Online 1, 74–76.
Biggers, J.D., 2001. Thoughts on embryo culture condition.
and continuous form, may cause serious harm and should be
Reprod. BioMed. Online 4, 30–38.
avoided. However, there are some situations where a well-
Biggers, J.D., Summers, M.C., 2008. Choosing a culture medium:
defined and properly applied stress may help gametes and making informed choices. Fertil. Steril. 90, 473–483.
embryos to increase their tolerance towards other stresses, Biggers, J.D., McGinnis, L.K., Lawitts, J.A., 2005. One-step versus
including those created during laboratory procedures. Thor- two-step culture of mouse preimplantation embryos: is there a
ough investigation of the molecular basis and safety of the difference? Human Reprod. 20, 3376–3384.
procedures are still required, but eventually HHP, osmotic Biggers, J.D., McGinnis, L.K., Summers, M.C., 2004. Dicrepancies
stress or other treatments with the same principle may im- between the effects of glutamine in cultures of preimplantation
prove the overall efficiency of assisted reproductive mouse embryos. Reprod. BioMed. Online 9, 70–73.
techniques. Biggers, J.D., McGinnis, L.K., Summers, M.C., 2006. Reply: one
step versus two-step culture of mouse preimplantation embryos.
Human Reprod. 21, 1936–1939.
Conclusion Booth, P.J., Humpherson, P.G., Watson, T.J., et al., 2005. Amino
acid depletion and appearance during porcine preimplantation
embryo development in vitro. Reproduction 130, 655–668.
Based on the impressive achievements during the past dec- Booth, P.J., Tan, S.J., Reipurth, R., et al., 2001. Simplification of
ade, there is an increasing, although rarely declared, doubt bovine somatic cell nuclear transfer by application of a zona-
about whether the present efficiency of in-vitro embryo cul- free manipulation technique. Cloning Stem Cells 3, 139–150.
466 G Vajta et al.

Carolan, C., Lonergan, P., Khatir, H., et al., 1996. In vitro A.J. (Eds.), A Laboratory Guide to the Mammalian Embryo.
production of bovine embryos using individual oocytes. Mol. Oxford University Press, Oxford, New York, pp. 41–61.
Reprod. Dev. 45, 145–150. Gardner, D.K., Lane, M., 2006. One-step versus two-step culture of
Chang, A.S., Moley, K.H., Wangler, M., et al., 2005. Association mouse preimplantation embryos. Human Reproduction 21,
between Beckwith–Wiedemann syndrome and assisted repro- 1935–1936.
ductive technology: a case series of 19 patients. Fertil. Steril. Gardner, D.K., Lane, M., 2007. Embryo culture systems. In:
83, 349–354. Gardner, D.K. (Ed.), In Vitro Fertilization: A Practical Approach.
Conaghan, J., Handyside, A.H., Winston, R.M., Leese, H.J., 1993. Informa Healthcare, New York, USA, pp. 221–282.
Effects of pyruvate and glucose on the development of human Gardner, D.K., Lane, M., Batt, P.A., 1993. Uptake and metabo-
preimplantation embryos in vitro. J. Reprod. Fertil. 99, 87–95. lism of pyruvate and glucose by individual sheep pre-attach-
Desai, N., Abdelhafez, F., Bedaiwy, M.A., et al., 2008. Live births ment embryos developed in vivo. Mol. Reprod. Develop. 36,
in poor prognosis IVF patients using a novel non-contact human 313–339.
endometrial co-culture system. Reprod. Biomed. Online 16, Gardner, D.K., Lane, M., Calderon, I., et al., 1996. Environment of
869–874. the preimplantation human embryo in vivo: Metabolite analysis
Deter, R.L., 1977. Quantitative morphological analysis of early of oviduct and uterine fluids and metabolism of cumulus cells.
mouse embryogenesis in vitro. J. Embryol. Exper. Morphol. 40, Fertil. Steril. 65, 349–353.
91–100. Ghanem, N., Hoelker, M., Phatsara, C., et al., 2009. Effect of well
Dominguez, F., Gadea, B., Mercader, A., et al., 2008. Embryologic in well culture of bovine embryos on gene expression profile.
outcome and secretome profile of implanted blastocysts Reprod., Fertil. Develop. 21, 190–191.
obtained after coculture in human endometrial epithelial cells Gopichandran, N., Leese, H.J., 2006. The effect of paracrine/
versus the sequential system. Fertil. Steril. 34, 45–56, Decem- autocrine interactions on the in vitro culture of bovine preim-
ber 3 [Epub ahead of print]. plantation embryos. Reproduction 131, 269–277.
Donnay, I., Van Langendonckt, A., Auquier, P., et al., 1997. Effects Hagemann, L.J., Weilert, L.L., Beaumont, S.E., et al., 1998.
of co-culture and embryo number on the in vitro development of Development of bovine embryos in single in vitro production
bovine embryos. Theriogenology 47, 1549–1561. (sIVP) system. Mol. Reprod. Develop. 51, 123–147.
Du, Y., Lin, L., Schmidt, M., Bøgh, I.B., et al., 2008b. High Holm, P., Booth, P.J., Schmidt, M.H., et al., 1999. High bovine
hydrostatic pressure treatment of porcine oocytes before blastocyst development in a static in vitro production system
handmade cloning improves developmental competence and using SOFaa medium supplemented with sodium citrate and
cryosurvival. Cloning Stem Cells 10, 325–330. myoinositol with or without serum. Theriogenology 52, 683–700.
Du, Y., Pribenszky, C.S., Molnár, M., et al., 2008a. High hydro- Houghton, F.D., Hawkhead, J.A., Humpherson, P.A., et al., 2002.
static pressure: a new way to improve in vitro developmental Non-invasive amino acid turnover predicts human embryo
competence of porcine matured oocytes after vitrification. developmental capacity. Human Reprod. 17, 999–1005.
Reproduction 135, 13–17. Hunter, R.H.F., 2005. The Fallopian tubes in domestic mammals:
Duranthon, V., Watson, A.J., Lonergan, P., 2008. Preimplantation how vital is their physiological activity? Reprod., Nutrition
embryo programming: transcription, epigenetics, and culture Develop. 45, 281–290.
environment. Reproduction 135, 141–150. Hunter, R.H.F., Bogh, I.B., Einter-Jensen, N., et al., 2000. Pre-
Ecker, D.J., Stein, P., Xu, Z., et al., 2004. Long-term effects of ovulatory graafian follicles are cooler than neighbouring stroma
culture of preimplantation mouse embryos on behaviour. Proc. in pig ovaries. Human Reprod. 15, 273–283.
National Acad. Sci. USA 101, 1595–1600. Hunter, R.H.F., Einer-Jensen, N., Greve, T., 2006. Presence and
Eckert, J., Pugh, P.A., Thompson, J.G., et al., 1999. Exogenous significance of temperature gradients among different ovarian
protein affects developmental competence and metabolic tissues. Microsc. Res. Tech. 69, 501–507.
activity of bovine preimplantation embryos in vitro. Reprod., Hunter, R.H.F., Nichol, R., 1986. A preovulatory temperature
Fertil. Develop. 10, 327–332. gradient between the isthmus and ampulla of pig oviducts during
Edwards, R.G., 1981. Test-tube babies. Nature 293, 253–256. the phase of sper storage. J. Reprod. Fertil. 77, 599–606.
Feltrin, C., Forell, F., dos Santos, L., et al., 2006. In vitro bovine Hunter, R.H.F., Nichol, R., Leese, H.J., 2003. Fallopian tube
embryo development after nuclear transfer by handmade microenvironments, gametes and early embryo development.
cloning using a modified WOW culture system. Reprod., Fertil. Havemeyer Found. Monogr. Ser. 10, 3–4.
Develop. 18, 126. Isom, S.C., Lai, L., Prather, R.S., et al., 2009. Heat shock of
Fernandez-Gonzalez, R., Moreira, P., Bilbao, A., et al., 2004. porcine zygotes immediately after oocyte activation increases
Long-term effect of in vitro culture of mouse embryos with viability. Mol. Reprod. Develop. 76, 548–554.
serum on mRNA expression of imprinting genes, development, Kattal, N., Cohen, J., Barmat, L.I., 2008. Role of coculture in human
and behaviour. Proc. National Acad. Sci. USA 101, 5880–5885. in vitro fertilization: a meta-analysis. Fertil. Steril. 90, 1069–1076.
Gandolfi, F., 1994. Autocrine, paracrine and environmental factors Khosla, S., Dean, W., Brown, D., et al., 2001a. Culture of
influencing embryonic development from zygote to blastocyst. preimplantation mouse embryos affects fetal development
Theriogenology 41, 95–100. and the expression of imprinted genes. Biol. Reprod. 64, 918–926.
Gardner, D.K., 2008. Dissection of culture media for embryos: the Khosla, S., Dean, W., Reik, W., et al., 2001b. Culture of
most important and less important components and character- preimplantation embryos and its long-term effects on gene
istics. Reprod., Fertil. Develop. 20, 9–18. expression and phenotype. Human Reprod. Update 7, 419–427.
Gardner, D.K., Lane, M., 1993. Amino acids and ammonium Kuo, Y.H., Pribenszky, C., Huang, S.Y., 2007. Higher litter size is
regulate mouse embryo development in culture. Biol. Reprod. achieved by the insemination of high hydrostatic pressure-
48, 377–385. treated frozen-thawed boar semen. In: Proceedings of the 6th
Gardner, D.K., Lane, M., 1997. Culture and selection of viable International Conference on Boar Semen Preservation, Alliston,
blastocysts: a feasible proposition for human IVF? Human Ontario, Canada, p. III–22.
Reprod. Update 3, 367–382. Kültz, D., 2003. Evolution of the cellular stress proteome: from
Gardner, D.K., Lane, M., 2003. Blastocyst transfer. Clin. Obstet. monophyletic origin to ubiquitous function. J. Exper. Biol. 206
Gynaecol. 46, 231–238. (Pt 18), 3119–3124.
Gardner, D.K., Lane, M., 2004. Culture of the mammalian Kültz, D., 2005. Molecular and evolutionary basis of the cellular
preimplantation embryo. In: Gardner, D.K., Lane, M., Watson, stress response. Annu. Rev. Physiol. 67, 225–257.
Improving embryo culture 467

Kwong, W.Y., Wild, A.E., Roberts, P., et al., 2000. Maternal McLaren, A., Biggers, J.D., 1958. Successful development and
undernutrition during the preimplantation period of rat devel- birth of mice cultivated in vitro as early as early embryos.
opment causes blastocyst abnormalities and programming of Nature 182, 877–878.
postnatal hypertension. Development 127, 4195–4202. Meintjes, M., Chantilis, S.J., Douglas, J.D., et al., 2009a. A
Lagutina, I., Lazzari, G., Duchi, R., et al., 2007. Comparative controlled randomized trial evaluating the effect of lowered
aspects of somatic cell nuclear transfer with conventional and incubator oxygen tension on live births in a predominantly
zona-free methods in cattle, horse, pig and sheep. Theriogen- blastocyst transfer program. Human Reprod. 24, 300–307.
ology 67, 90–98. Meintjes, M., Chantilis, S.J., Ward, D.C., 2009b. A randomized
Lane, M., Gardner, D.K., 1992. Effect of incubation volume and controlled study of human serum albumin and serum substitute
embryo density on the development and viability of mouse supplement as protein supplements for IVF culture and the
embryos in vitro. Human Reprod. 7, 558–562. effect on live birth rates. Human Reprod. 24, 782–799.
Lane, M., Gardner, D.K., 1994a. Increase in postimplantation Mendes, P.M., 2008. Stimuli-responsive surfaces for bio-applica-
development of cultured mouse embryos by amino acids and tions. Chem. Soc. Rev. 37, 2512–2529.
induction of fetal retardation and exencephaly by ammonium Ménézo, Y.J., Hérubel, F., 2002. Mouse and bovine models for
ions. J. Reprod. Fertil. 102, 305–312. human IVF. Reprod. BioMed. Online 4, 170–175.
Lane, M., Gardner, D.K., 1994b. Amino acids increase mouse Mercader, A., Valbuena, D., Simón, C., 2006. Human embryo
embryo viability. Theriogenology 41, 233. culture. Methods Enzymol. 420, 3–18.
Lane, M., Gardner, D.K., 2001. Inhibiting 3-phosphoglycerate Miller, A.O., Menozzi, F.D., Dubois, D., 1989. Microbeads and
kinase by EDTA stimulates the development of the cleavage anchorage-dependent eukaryotic cells: the beginning of a new era
stage mouse embryos. Mol. Reprod. Develop. 60, 233–240. in biotechnology. Adv. Biochem. Eng./Biotechnol. 39, 73–95.
Lane, M., Gardner, D.K., 2003. Ammonium induces aberrant Mortimer, D., 1986. Elaboration of a new culture medium for
blastocyst differentiation, metabolism, pH regulation, gene physiological studies on human sperm motility and capacitation.
expression and subsequently alters fetal development in the Human Reprod. 1, 247–250.
mouse. Biol. Reprod. 69, 1109–1117. Mortimer, D., 2001. Human blastocyst development media. Human
Lane, M., Gardner, D.K., 2007. Embryo culture medium: which is Reprod. 16, 2725.
the best? Best Practice Res. Clin. Obst. Gynaecol. 21, 83–100. Nagao, Y., Saeki, K., Hoshi, M., et al., 1994. Effects of oxygen
Lawrence, L.T., Moley, K.H., 2008. Epigenetics and assisted concentration and oviductal epithelial tissue on the develop-
reproductive technologies: human imprinting syndromes. Sem- ment of in vitro matured and fertilized bovine oocytes cultured
inars Reprod. Med. 26, 143–152. in protein-free medium. Theriogenology 41, 681–687.
Leach, R.E., Stachecki, J.J., Armant, D.R., 1993. Development of Oh, S.J., Gong, S.P., Lee, S.T., et al., 2007. Light intensity and
in vitro fertilized mouse embryos exposed to ethanol during the wavelength during embryo manipulation are important factors
preimplantation period: accelerated embryogenesis at subtoxic for maintaining viability of preimplantation embryos in vitro.
levels. Teratology 47, 57–64. Fertil. Steril. 88 (4 Suppl.), 1150–1157.
Lee, E.S., Pugh, P.A., Allen, N., 1998. [5–3H]-glucose and [1–14C]- Oliver, M.H., Jacquiery, A.L., Bloomfield, F.H., 2007. The effects
pyruvate utilization by fresh and frozen-thawed Day 7 IVP bovine of maternal nutrition around the time of conception on the
blastocysts cultured in PVA-or BSA-supplemented medium. health of the offspring. J. Reprod. Fertil. 64 (Suppl.), 397–410.
Theriogenology 49, 233. O’Neill, C., 1997. Evidence for the requirement of autocrine
Leese, H.J., 1998. Human embryo culture: back to nature. J. growth factors for development of mouse preimplantation
Assisted Reprod. Genetics 15, 466–468. embryos in vitro. Biol. Reprod. 56, 229–237.
Leese, H.J., 2002. Quiet please, do not disturb: a hypothesis of O’Neill, C., 2008. The potential roles for embryotrophic ligands in
embryo metabolism and viability. Bioessays 24, 845–849. preimplantation embryo development. Human Reprod. Update
Leese, H.J., 2003. What does an embryo need? Human Fertil. 14, 275–288.
(Camb.) 6, 180–185. Otsuki, J., Nagai, Y., Chiba, K., 2007. Peroxidation of mineral oil
Leese, H.J., Barton, A.M., 1984. Pyruvate and glucose uptake by used in droplet culture is detrimental to fertilization and embryo
mouse ova and preimplantation embryos. J. Reprod. Fertil. 72, development. Fertil. Steril. 88, 741–743.
9–13. Otsuki, J., Nagai, Y., Chiba, K., 2009. Damage of embryo
Leese, H.J., Conaghan, J., Martin, K.L., et al., 1993. Early human development caused by peroxidized mineral oil and its associ-
embryo metabolism. Bioessays 15, 259–264. ation with albumin in culture. Fertil. Steril. 91, 1745–1749.
Leese, H.J., Baumann, C.G., Brison, D.R., et al., 2008a. Metab- Paoloni-Giacobino, A., 2007. Genetic and epigenetic risks of ART.
olism of the viable mammalian embryo: quietness revisited. Mol. Fertil. Steril. 88, 761–762.
Human Reprod. 14, 667–672. Parikh, F.R., Nadkarni, S.G., Naik, N.J., et al., 2006. Cumulus
Leese, H.J., Hugentobler, S.A., Gray, S., 2008b. Female repro- coculture and cumulus-aided embryo transfer increases
ductive tract fluids: composition, mechanism of formation and pregnancy rates in patients undergoing in vitro fertilization.
potential role in the developmental origins of health and Fertil. Steril. 86, 839–847.
disease. Reprod., Fertil. Develop. 20, 1–8. Peura, T.T., Vajta, G., 2003. A comparison of established and new
Leese, H.J., Tay, J.I., Reischl, J., et al., 2001. Formation of approaches in ovine and bovine nuclear transfer. Cloning Stem
fallopian tubal fluid: role of a neglected epithelium. Reproduc- Cells 5, 257–277.
tion 121, 339–346. Pool, T.B., 2002. An update on embryo culture for human assisted
Lin, L., Du, Y., Liu, Y., et al., 2009a. Elevated NaCl concentration reproductive technology: media, performance, and safety sem-
improves cryotolerance and developmental competence of inars. Reprod. Med. 23, 309–318.
porcine oocytes. Reprod. BioMed. Online 18, 360–366. Pool, T.B., 2005. Recent advances in the production of viable
Lin, L., Kragh, P.M., Purup, S., et al., 2009b. Osmotic stress human embryos in vitro. Reprod. BioMed. Online 4, 294–302.
induced by sodium chloride sucrose or trehalose improves Pribenszky, C., Molnár, M., Cseh, S., et al., 2005a. Improving post-
cryotolerance and developmental competence of porcine thaw survival of cryopreserved mouse blastocysts by hydrostatic
oocytes. Reprod., Fertil. Develop. 21, 338–344. pressure challenge. Animal Reprod. Sci. 87, 143–150.
Lonergan, P., Fair, T., Corcoran, D., et al., 2006. Effect of culture Pribenszky, C., Molnár, M., Horváth, A., 2005b. Hydrostatic
environment on gene expression and developmental character- pressure induced increase in post-thaw motility of frozen boar
istics in IVF-derived embryos. Theriogenology 65, 137–152. spermatozoa. Reprod., Fertil. Develop. 18, 162–163.
468 G Vajta et al.

Pribenszky, C., Molnár, M., Ulrich, P., et al., 2005c. Pressure Sinclair, K.D., 2008. Assisted reproductive technologies and
assisted cryopreservation: a novel possibility for IVP bovine pregnancy outcomes: mechanistic insights from animal studies.
blastocyst cryopreservation. Reprod. Domestic Animals 40, 338. Seminars Reprod. Med. 26, 153–161.
Pribenszky, C., Molnár, M., Horváth, A., et al., 2007a. Improved Smith, A.U., 1951. Fertilisation in vitro of the mammalian egg. In:
post-thaw motility, viability and fertility are achieved by Biochemical Society Symposia (Cambridge), vol. 7 (cit. Bavister,
hydrostatic pressure treated bull semen. Reprod., Fertil. 2002), pp. 3–10.
Develop. 19, 181–182. Smith, G.D., Takayama, S., 2007. Gamete and embryo isolation
Pribenszky, C., Siquiera, E., Molnár, M., et al., 2007b. Improved and culture with microfluidics. Theriogenology 68S, S190–S195.
post-warming developmental competence of open pulled straw- Steeves, T.E., Gardner, D.K., 1999. Temporal and differential
vitrified in vitro-produced bovine blastocysts by sublethal effects of amino acids on bovine embryo development in
hydrostatic pressure pre-treatment. Reprod., Fertil. Develop. culture. Biol. Reprod. 61, 731–740.
20, 125. Summers, M.C., Biggers, J.D., 2003. Chemically defined media and
Pribenszky, C., Du, Y., Molnár, M., et al., 2008a. Increased stress culture of mammalian preimplantation embryos: historical
tolerance of pig oocytes after high hydrostatic pressure treat- perspective and current issues. Human Reprod. Update 9,
ment. Animal Reprod. Sci. 106, 200–207. 557–582.
Pribenszky, C., Molnár, M., Kútvölgyi, G., et al., 2008b. Sublethal Tay, J.L., Rutheford, A.J., Killick, S.R., et al., 1997. Human tubal
stress treatment of fresh boar semen with high hydrostatic fluid: production, nutrient composition and response to adren-
pressure, inserted into the routine insemination procedure may ergic agents. Human Reprod. 12, 2451–2456.
improve average litter size by 1 piglet. Reprod. Domestic Thompson, J.G., 1996. Defining the requirements for bovine
Animals 43, 108. embryo culture. Theriogenology 45, 27–40.
Quinn, P., 1995. Enhanced results in mouse and human embryo Thompson, J.G., 2000. In vitro culture and embryo metabolism of
culture using a modified human tubal fluid medium lacking cattle and sheep embryos – a decade of achievement. Animal
glucose and phosphate. J. Assisted Reprod. Gen. 12, 97–105. Reprod. Sci. 60–61, 263–275.
Quinn, P., 2000. Review of media used in ART laboratories. J. Thompson, J.G., 2007. Culture without the Petri dish. Theriogen-
Androl. 21, 610–615. ology 67, 16–20.
Quinn, P., 2004. The development and impact of culture media for Thompson, J.G., Peterson, A.J., 2000. Bovine embryo culture
assisted reproductive technologies. Fertil. Steril. 81, 27–29. in vitro: new developments and post-transfer consequences.
Quinn, P., Horstman, F.C., 1998. Is the mouse a good model Human Reprod. 15 (Suppl. 5), 59–67.
for the human with respect to the development of the Thompson, J.G., Gardner, D.K., Pugh, P.A., et al., 1995. Lamb
preimplantation embryo in vitro? Human Reprod. 13 (Suppl. birth weight is affected by culture system utilized during in vitro
4), 173–183. pre-elongation development of ovine embryos. Biol. Reprod. 53,
Quinn, P., Kerin, J.F., Warnes, G.M., 1985. Improved pregnancy 1385–1391.
rate in human in vitro fertilization with the use of a medium Thompson, J.G., Mitchell, M., Kind, K.L., 2007. Embryo culture and
based on the composition of human tubal fluid. Fertil. Steril. 44, long-term consequences. Reprod. Fertil. Develop. 19, 43–52.
493–498. Thouas, G.A., Jones, G.M., Trounson, A.O., 2003. The ‘GO’
Rahman, P.K., Pasirayi, G., Auger, V., et al., 2009. Development system–a novel method of microculture for in vitro develop-
of a simple and low cost microbioreactor for high-throughput ment of mouse zygotes to the blastocyst stage. Reproduction
bioprocessing. Biotechnol. Lett. 31, 209–214. 126, 161–169.
Richter, K.S., 2008. The importance of growth factors for Trounson, A., Conti, A., 1982. Research in human in-vitro fertil-
preimplantation embryo development and in-vitro culture. Curr. isation and embryo transfer. Brit. Med. J. (Clin. Res. Ed.). 285,
Opin. Obst. Gynecol. 20, 292–304. 244–248.
Rieger, D., Schimmel, T., Cohen, J., et al., 2007. Comparison of Trounson, A., Pushett, D., Maclellan, L.J., et al., 1994. Current
GPS and standard dishes for embryo culture: set-up and status of IVM/IVF and embryo culture in humans and farm
observation times, and embryo development. In: Proceedings animals. Theriogenology 41, 57–66.
of the 14th World Congress on IVF and 3rd World Congress on Vajta, G., Alexopoulos, N.I., Callesen, H., 2004. Rapid growth and
IVM, Montreal, p. 1202. elongation of bovine blastocysts in vitro in a three-dimensional
Roh, S., Choi, Y.-J., Min, B.-M., 2008. A novel microtube culture gel system. Theriogenology 62, 1253–1263.
system that enhances the in vitro development of parthenoge- Vajta, G., Holm, P., Greve, T., et al., 1997. The submarine
netic murine embryos. Theriogenology 69, 262–267. incubation system, a new tool for in vitro embryo culture. a
Rooke, J.A., McEvoy, T.G., Ashworth, C.J., 2007. Ovine fetal technique report. Theriogenology 48, 1379–1385.
development is more sensitive to perturbation by the presence Vajta, G., Holm, P., Kuwayama, M., et al., 1998. Open Pulled Straw
of serum in embryo culture before rather than after compaction. (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova
Theriogenology 67, 639–647. and embryos. Mol. Reprod. Develop. 51, 53–58.
Schini, S.A., Bavister, B.D., 1988. Two-cell block to development Vajta, G., Ko }rösi, T., Du, Y., et al., 2008. The Well-of-the-Well
of cultured hamster embryos is caused by phosphate and system: an efficient approach to improve embryo development.
glucose. Biol. Reprod. 39, 1183–1192. Reprod. BioMed. Online 17, 73–81.
Selye, H., 1998. A syndrome produced by diverse nocuous agents. Vajta, G., Kragh, P.M., Mtango, N.R., et al., 2005. Handmade
1936. J. Neuropsych. Clin. Neurosci. 10, 230–231. cloning approach: potentials and limitations. Reprod., Fertil.
Sepúlveda, S., Garcia, J., Arriaga, E., et al., 2008. In vitro Develop. 17, 97–112.
development and pregnancy outcomes for human embryos Vajta, G., Lewis, I.M., Hyttel, P., et al., 2001. Somatic cell
cultured in either a single medium or in a sequential media cloning without micromanipulators. Cloning 3, 89–95.
system. Fertil. Steril. 91, 1765–1770. Vajta, G., Peura, T.T., Holm, P., et al., 2000. New method
Shettles, L.B., 1953. Observations on human follicular and tubal for culture of zona-included and zone-free embryos: the Well
ova. Am. J. Obst. Gynecol. 66, 235–247. of the Well (WOW) system. Mol. Reprod. Develop. 55,
Silver L: Human-animal Chimeras: From Mythology To Biotechnol- 256–264.
ogy. In Scientific Blogging, Science 2.0, 2007. <http://www.sci- Vajta, G., Zhang, Y., Macháty, Z., 2007. Somatic cell nuclear
entificblogging.com/lee_silver/human_animal_chimeras_from_ transfer in pig: recent achievements and future possibilities.
mythology_to_biotechnology>. Reprod., Fertil. Develop. 19, 403–423.
Improving embryo culture 469

Van Wagtendonk-de Leeuw, A.M., Aerts, B.j., den Daas, J.H., Wood, S.A., Allen, N.D., Rossant, J., et al., 1993. Non-injection
1998. Abnormal offspring following in vitro production of bovine methods for the production of embryonic stem cell-embryo
preimplantation embryos: a field study. Theriogenology 49, chimaeras. Nature 365, 87–89.
883–894. Wrenzycki, C., Herrmann, D., Lucas-Hahn, A., et al., 2005.
Virant-Klun, I., Tomazevic, T., Vrtacnik-Bogal, E., et al., 2006. Messenger RNA expression patterns in bovine embryos derived
Increased ammonium in culture medium reduces the develop- from in vitro procedures and their implications for development.
ment of human embryos to the blastocyst stage. Fertil. Steril. Reprod., Fertil. Develop. 17, 23–35.
85, 526–528. Yoshioka, K., Suzuki, C., Tanaka, A., et al., 2000. Birth of piglets
Vodicka, P., Smetana Jr, K., Dvoránková, B., et al., 2005. The derived from porcine zygotes cultured in a chemically defined
miniature pig as an animal model in biomedical research. Ann. medium. Biol. Reprod. 66, 112–119.
New York Acad. Sci. 1049, 161–171. Yovich, J., Grudzinskas, G., 1990. The Management of Infertility: A
Voelkel, S.A., Hu, Y.X., 1992. Effect of gas atmosphere on the Manual of Gamete Handling Procedures. Heinemann Medical
development of one-cell bovine embryos in two culture systems. Books, Oxford.
Theriogenology 37, 1117–1131. Zander, D.L., Thompson, J.G., Lane, M., 2006. Perturbations in
Wemekamp-Kamphuis, H.H., Karatzas, A.K., et al., 2002. mouse embryo development and viability caused by ammonium
Enhanced levels of cold shock proteins in Listeria Monocitygenes are more severe after exposure at the cleavage stages. Biol.
LO28 upon exposure to low temperature and high hydrostatic Reprod. 74, 288–294.
pressure. Appl. Environ. Microbiol. 68, 456–463.
Wernersson, R., Schierup, M.H., Jørgensen. F.G., et al., 2005. Pigs Declaration: Gabor Vajta is a minority shareholder in the company
in sequence space: a 0.66X coverage pig genome survey based on that produces tools for HHP and WOW studies. Other authors
shotgun sequencing. BMC Genomics 6, p. 70. doi: 10.1186/1471– declare no commercial interest.
21 64–6-70.
Whitten, W.K., 1956. Culture of tubal mouse ova. Nature 177, 96–97. Received 19 May 2009; refereed 20 July 2009; accepted 9 December
Whitten, W.K., 1957. Culture of tubal ova. Nature 179, 1081–1082. 2009.