Human assisted conception: a cautionary tale.
Lessons from domestic animals
HJ.Leese 14 ,1.Donnay 2 and J.G.Thompson3
department of Biology, University of York, PO Box 373, York YO1 5YW,
UK, 2Sciences Veterinaires, Universite Catholique de Louvain, Belgium,
and 3Ag Research, Ruakura, PB 3123 Hamilton, New Zealand
4
To whom correspondence should be addressed
A variety of embryo-based technologies used in farm animal reproduction,
including embryo culture, nuclear transfer, embryo-somatic cell co-culture
and asynchronous embryo transfer can lead to the production of large
offspring; the so-called large calf/lamb syndrome. In some cases, abnormalities in the fetus and newborn are apparent. The nature of these associations
is explored with emphasis on the biological differences between in-vivo- and
in-vitro-produced embryos. A unifying framework and research programme
aimed at explaining anomalies in early embryo development is then proposed
in terms of the response of somatic cells and embryos to cellular stress. The
review concludes with a caution against developments in assisted conception
technologies, in man and domestic animals, being determined too much by
the needs of commerce at the expense of research on the molecular,
biochemical and physiological basis of early mammalian development.
Key words: assisted conception/review
Introduction
Behind the headlines announcing new techniques in human assisted conception,
there are findings from work on the domestic species which should make us
pause and reflect. In these species, a variety of techniques used in farm animal
reproduction can lead to the production of large offspring after embryo transfer;
the so-called large calf/lamb syndrome. In some cases, abnormalities in the fetus
or newborn are also apparent.
Techniques which can predispose to fetal oversize include: nuclear transfer
(Wilson et al, 1995); embryo culture (Walker et al, 1992a,b; Thompson et al,
1995); embryo-somatic cell co-culture (Farm and Farin, 1995) and asynchronous
embryo transfer (Wilmut and Sales, 1981).
These phenomena threaten to limit the application of the 'new genetics' to the
improvement of agricultural production, and could have implications for human
assisted conception, but their time of onset in the early conceptus and the
underlying mechanisms are unknown.
We begin this review by considering the history of these in-vitro-induced
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© European Society for Human Reproduction and Embryology Human Reproduction Volume 13 Supplement 4 1998
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problems in the sheep before describing related problems in the cow. We then
discuss possible explanations for these fetal anomalies. This leads us to search
for a unifying mechanism in terms of the response of somatic cells to stress. In
the light of this, we put forward a model of the molecular, biochemical and
physiological origins of anomalies in the early embryo. The review briefly
considers the economic and technological factors that determine the direction of
science in this area and ends with a plea for a vigorous programme of basic
research on the response of early embryos to cellular stress.
Culture-induced abnormalities in fetal and neonatal sheep development
Compared with other species, particularly the mouse, progress in the development
of culture systems for the early embryos of domestic ruminants has mostly
occurred over the past decade. Earlier workers found these embryos to be difficult
to grow under in-vitro conditions, as discussed in the thorough review by Wright
and Bondioli (1981). These authors concluded that the culture-induced block to
development at the 8-16-cell stage in sheep and cattle had not yet been resolved
despite occasional reports documenting various degrees of success in the
development of 1- and 2-cell stage embryos into blastocysts. Two such reports
(Tervit et al, 1972, 1974) from the University of Cambridge, described a new
medium, synthetic oviduct fluid (SOF), a formulation loosely based on the
composition of sheep oviduct fluid. However, widespread use of this medium
did not follow, most likely due to other laboratories being unable to repeat the
success reported by Tervit et al (1972, 1974). This may have been due not to
the medium as such, but to other factors such as the need to use the correct gas
mixture and ensure the purity of the medium components. Nevertheless, most
laboratories with an interest in culturing the embryos of domestic ruminants
appeared to abandon the use of this formulation. Instead, co-culture techniques,
such as that described by Gandolfi and Moor (1987) were adopted, especially
when linked with methods to produce embryos following in-vitro maturation and
in-vitro fertilization (IVF).
However, one group at the University of Adelaide persisted with the development and application of the SOF formulation. In a rather bizarre twist of fate,
probably due to the fact that this group was also involved in a human IVF
programme and thus had access to a ready supply of human serum (HS), it was
reported that supplementing the SOF medium with human serum (20% v/v),
compared with other sources of protein and sera, improved the proportion of
1-cell zygotes developing to the blastocyst stage after 5 days of culture (Quinn
et al, 1984; Walker et al, 1988). Levels of development of sheep 1- and 2-cell
embryos recovered from ovulation-stimulated ewes, were consistently high
(>80%). Application of this system to bovine embryo culture was also successfully achieved (McLaughlin et al, 1990; Fukui et al, 1991). However, following
the transfer of ovine embryos cultured in this medium, a proportion of the
resulting lambs were heavy at birth, with some extreme weights recorded,
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H.J.Leese, I.Donnay and J.G.Thompson
compared with naturally mated or artificially inseminated controls. The overall
shift in birth weight was 20% on average and the distribution was skewed
towards heavy animals (Walker et al, 1992a,b). Gestation length was also
lengthened, on average, by 2 days, but did not account for the change in birth
weight (Walker et al, 1992a,b). A high incidence of neonatal death, dystocia
and limb abnormalities were also described in the lambs born (Walker et al,
1992a,b, 1996). Post-natal growth, at least up to 6 months of age, has since been
reported to be greater in lambs born following embryo culture in the presence
of serum compared with naturally-bred controls (Brown and Radziewic, 1996)
and shifts in the allometry of fetal heart and liver weights following transfer of
embryos derived either by co-culture, or culture in SOF + HS, have been
observed in day 61 fetuses (Sinclair et al., 1997). However, an increase in fetal
weight was observed only for fetuses derived from co-cultured embryos.
Ovine embryo morphology and physiology in vivo and in vitro
Blastocyst-stage ovine embryos resulting from culture in vitro in medium with
human serum have a number of morphological differences to those derived
in vivo. Most notably, in-vitro produced (IVP) embryos blastulate earlier (by ~24
h) than those developing in vivo (Walker et al, 1992a,b) and the level of
compaction observed at the morula stage is significantly less for embryos cultured
in serum-supplemented medium (Thompson, 1997). In addition, the degree of
cytoplasmic fragmentation is greater in these cultured embryos than in their invivo counterparts (Walker et al, 1988, 1992a,b). Premature blastulation and
fragmentation may lead to increased birth weights, perhaps by a shift in the ratio
between inner cell mass (ICM) and trophectoderm (TE) cells, which could, in
turn, lead either to large placentation or fetal oversize.
Embryos cultured in SOF + HS appear to be dark when observed under a
dissecting microscope and are less reflective of incident light compared with
serum-free cultured or in-vivo-derived embryos (Dorland et al, 1994; Gardner
et al, 1994; Thompson et al, 1995). This is due to the accumulation of lipid
droplets within the cytoplasm (Dorland et al, 1994; Thompson et al, 1995). At
least some of this lipid is osmophilic, representing unsaturated forms, and is
probably derived from triglycerides contained in serum lipoproteins derived from
serum (Thompson et al, 1995). Apart from possible metabolic effects, this lipid
accumulation leads to a dramatic increase in cell volume and poses potential
osmotic problems for the embryo. The level of glucose oxidation is lower for
in-vitro cultured embryos than those derived in vivo (Thompson et al, 1991).
Pyruvate oxidation is essentially the same on a per embryo basis between in-vivoderived blastocysts and those following culture in SOF + HS. However, at a
cellular level, pyruvate oxidation is markedly higher for in-vitro- than
in-vivo-derived embryos, because of the reduced cell number in the case of the
former (Thompson et al, 1996).
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Table I. Mean birth weight (± SEM) of lambs produced from embryos cultured in synthetic
oviduct fluid (SOF). (Data taken from Thompson et al, 1995)
Culture medium
Birth weight (± SEM) (kg)
SOF + HS
SOFaa BSA
Control
4.2 ± 0.2a
3.5 :± 0.2
3.4 ± 0.2
SOF + HS = SOF supplemented with 20% human serum; SOFaa BSA = SOF supplemented
with pooled non-essential and essential amino acids.
Significantly heavier than SOFaaBSA and controls (P < 0.05) Difference still significant (P <
0.05) after adjustment for differences in gestation length.
Cell and serum-free embryo culture systems
The development of a serum- and somatic cell-free culture system for ruminant
embryos came about from the desire to define cellular requirements during
early development without the confounding influence of components whose
composition is undefined. A system which effectively matched the performance
of human serum-supplemented media to support ovine embryo development was
eventually achieved through knowledge of the benefits of pooled amino acid
addition (Gardner et ah, 1994), the negative effect of ammonia, derived both
from metabolism and via degradation of amino acids (Gardner et ah, 1993, 1994)
and the positive effect of grouping embryos in relatively small volumes of
medium (Paria and Dey, 1990; Lane and Gardner, 1992; Gardner et ah, 1994),
thereby enhancing the activity of autocrine growth factors (Thibodeaux et ah,
1995). This greater understanding led to an adjustment in the original SOF
formulation; namely, the inclusion of pooled non-essential and essential amino
acids (SOFaaBSA) and the replacement of medium every 48 h during the culture
period (Gardner, 1994; Gardner et ah, 1994). Under such conditions, at an
appropriate embryo density, development to the blastocyst stage of 1- and 2-cell
sheep zygotes of <90% has been reported, with comparable cell numbers to
in-vivo-derived blastocysts of similar age and stage (Gardner et ah, 1994).
Moreover, the transfer of such embryos led to lambs with similar birth weights
to those bred naturally and which were significantly below those produced from
embryos cultured in SOF supplemented with human serum (Thompson et ah,
1995) and Table I. This, and other data from the mouse (Lane and Gardner,
1994) demonstrate that it is not the period of in-vitro culture itself which imposes
these abnormalities but the system used (i.e. medium components and physical
culture conditions), which affects developmental outcome.
Conclusions
Most of the abnormalities discussed above are a function of culture in serum
and/or the presence of somatic cells. Abnormalities are either absent or greatly
reduced in embryos cultured under serum-and cell-free conditions (i.e. in
SOFaaBSA medium)
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Table II. Influence of technique on mean gestation length, birth weights, course of parturition
(dystocia scores), Caesarean sections and perinatal death of Holstein Friesian calves on the same
120 farms. (Taken from Kruip and den Daas, 1997)
Technique
No. of
calves
Gestation
length (days)
Birth
weight (kg)
Dystocia
scores
AI
ET
IVP
1160
45
251
281.3 ± 0.2
283.5 ± 0.8
283.9 ± 0.4
42.8 ± 0.2
42.7 ± 0.8
46.2 ± 0.5
2.44 ± 0.04
2.74 ± 0.21
3.05 ±0.11
Caesarean Perinatal
section (%) death
0.9
13.0
6.1 ± 0.6
6.6 ± 0.6
14.4 ± 2.3
AI = artificial insemination; ET = conventional embryo transfer; IVP = in-vitro produced
embryos.
Bovine embryo development and the large calf syndrome
Non-surgical transfers of bovine IVP embryos, and of embryos cloned by nuclear
transfer (NT) can result in prolonged pregnancies and offspring with undesirably
high birth weights (Table II). Not all offspring are affected (Walker et al, 1996);
Kruip and den Daas (1997), in a large retrospective epidemiological study on
different cattle breeds, worldwide, described the increase in birthweight and
gestation length as a general shift in population means. The large calf syndrome
does not appear to be heritable (Walker et al, 1996), but its increased incidence
has important implications for animal health and welfare, as well as affecting
the commercial acceptance of IVP and NT techniques (Schernthaner et al, 1991 \
Kruip and den Daas, 1997).
Delay in the return to oestrus after unsuccessful transfer is a feature of IVP
embryo transfer. This indicates that such embryos can induce lengthening of the
corpus luteum lifespan with associated failures in embryogenesis or implantation.
Abortion rates of 9-47% during the first and second trimesters have been reported
(Looney et al, 1994; Van Soom et al, 1994; Wurth et al, 1994; Reinders et al,
1995; Farin and Farin, 1995) with anomalies in placental size and the number
of cotyledons.
There are higher incidences of dystocia, perinatal loss and anomalies (Table
II). These include heart failures, double muscles, hydroallantois (Hasler et al,
1995), leg and joint problems, large organs (Garry et al, 1996), limb deformities
(Wilson et al (1995) and cerebellar hypoplasia (Schmidt et al, 1996) which
might explain the reduced viability and higher percentage of perinatal death. IVP
and NT calves also showed a greater susceptibility to infectious diseases (Penny
et al, 1995; Schmidt et al, 1996; Garry et al, 1996).
Bovine embryos used in NT are produced in vivo or by IVM/IVF and then
cultured in different ways until transfer to recipients at the morula/blastocyst
stage (day 8 post-insemination). It is likely that the problems associated with
NT and IVP calves are due, as in the sheep, to the conditions of culture and/or
transfer but we cannot exclude alternative origins of the syndrome.
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Bovine embryo morphology and physiology in vivo and in vitro.
Farin and Farin (1995) examined the outcome of pregnancies following the
transfer of in-vitro and in-vivo-derived bovine embryos. The in-vitro-derived
embryos were co-cultured in TCM, 199 with bovine oviductal epithelial cells in
the presence of 10% oestrous cow serum, a system widely used for producing
cattle embryos. Significant differences between the two types of embryo were
observed after transfer: IVP embryos of the same morphological grade as invivo controls showed an increased rate of early embryonic death, increased fetal
weight at 7 months' gestation and disproportionate skeletal measurements. Longbone lengths and heart girth and weights were also increased. However, the
weights of the visceral organs on a body weight basis were not affected, implying
that the increased body weight may have resulted from increased bone density,
muscle mass, fat deposition, fluid volume, or some combination of these factors.
The number of placentomes on a body weight basis was reduced.
Pregnancy-associated proteins (bPAG and bPSPB) are secreted by binucleate
trophoblastic cells in the bovine and can be used as an indicator of placental
function. Schmidt et al. (1996) reported that bPAG profiles for heifers carrying
fetuses resulting from artificial insemination or from the transfer of in-vivo and
in-vitro produced embryos were identical (Schmidt et al., 1996). In this study,
few fetal losses were observed and the IVP calves were not overweight. However
the incidence of stillbirths and of post-natal calf mortality were increased in the
IVP group. The in-vitro embryos had been co-cultured with bovine oviduct
epithelial cells in B2 medium and transferred freshly. In another study where
IVP calves were overweight, bPSPB concentrations were higher in the IVP
pregnancies than in those established following multiple ovulation and embryo
transfer (MOET) or by artificial insemination, but effects were confounded by
fetal genotype (Vasques et al., 1995). Nevertheless the results of this last study
suggested that cows bearing larger calves produced significantly higher amounts
of bPSPB, which could indicate an oversized or overactive placenta.
With regard to NT, the discrepancy in growth between calves arising from the
same clone was confined to development in utero, as body size variation decreased
substantially by the time of weaning (Wilson et al., 1995). Willadsen et al.
(1991) and Garry et al. (1996) reported a high incidence of joint and limb
abnormalities in NT calves. This might be linked directly to the oversize of the
fetuses, which are cramped in utero. Most calves derived from NT did not suckle
well and most needed assistance for a long period postpartum. Wilson et al. (1995)
reported that postpartum weakness, hypothermia, hypoxaemia, hypoglycaemia and
metabolic acidosis were present in the majority of 40 NT calves suggesting a
defect in energy metabolism. In support of this proposition, there were reduced
concentrations of plasma thyroxine and T3 as well as high insulin concentrations,
compared with data from reference calves. After reviewing the literature from
other species, including the human, the authors suggested that the metabolic
problems observed following NT could be due to adaptations made by the
conceptus to abnormal placental function.
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Possible mechanisms of fetal anomalies arising during embryo
development
Embryo culture conditions or manipulations are known to affect several parameters
including the kinetics of development, cell allocation to the ICM and TE, embryo
metabolism and genomic expression. Those changes can directly or indirectly be
related to the large calf syndrome. Even embryos cultured in vivo in ligated
sheep oviducts can lead to overweight calves, at least after NT (Wilson
etal, 1995),
Kinetics of development
The kinetics of embryo development are affected by the system of culture,
blastulation appearing earlier in co-culture or in culture in semi-defined conditions
with fetal calf serum, than in defined conditions (Van Langendonckt et al, 1997).
Variations between co-culture systems have also been observed (Donnay et al,
1997) as well as following the use of different media to co-culture embryos with
the same somatic cells (Semple et al, 1993; Hasler et al, 1995; Van Soom et al,
1996). Alterations in the kinetics of development seem to appear after the onset
of genomic expression (Van Langendonckt et al, 1997).
It has also been shown that delayed blastocysts, appearing after 8 or more
days of culture, are of lower quality and viability after transfer than those formed
on day 7 (Hasler et al, 1995). Moreover, early cleaved embryos have a better
chance of becoming viable blastocysts (Greve et al, 1992; Grisart et al, 1994;
Van Soom et al, 1997). However, this does not imply that a culture system
ensuring faster development is superior (Van Langendonckt et al, 1997) and in
the bovine, comparisons of physiological timing are difficult to establish due to
a lack of data from unstimulated cows. Alterations in the kinetics of development
are also related to the allocation of the cells to the ICM and TE and can be part
of a cascade leading to the large calf syndrome.
Cell allocation to ICM and TE
IVP bovine blastocysts are often characterized by a lower, more variable, ratio
of ICM/TE cells than those derived in vivo (Du et al, 1996). This variability
depends on the culture system used (Van Soom et al, 1996). One explanation
could be differences in the compaction process which has been demonstrated to
be less pronounced in IVP than in in-vivo embryos (Greve et al, 1993; Van
Soom et al, 1997). The establishment of the gap junction seal depends on the
medium used for embryo culture (Van Soom et al, 1996). The altered compaction
observed in IVP embryos can be reversed; both in-vivo embryos collected at the
1- or 2-cell stage and cultured in vitro up to the blastocyst stage and IVP embryos
cultured in ligated sheep oviducts, showed a prolonged, tight compaction phase
which postponed their morula-blastocyst transition by one day compared with
their IVM/IVF/IVC counterparts (Greve et al, 1993; Van Soom, 1996). Prolonga190
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tion of the tight morula phase, seems to allow more cells to be allocated to the
ICM (Van Soom, 1996).
Blastocysts with a small, or in some cases, absent, inner cell mass cannot be
distinguished from normal embryos and are able to hatch (Avery et al, 1995;
Van Soom et al, 1997). When too few ICM cells are present, such embryos will
cease their development but can cause a delayed return to oestrus after transfer
of IVP embryos, through the secretion of the luteotrophic bovine trophoblast
protein 1 by TE cells (Farin et al, 1992). Thus, a decreased ICM/TE ratio could
lead to an imbalance in placental development which could explain some of the
features of the large calf syndrome.
Genomic expression
Embryo manipulation at an early stage could affect the transcription of one or
more genes associated with development. The problems could arise from the
systems of culture during oocyte maturation, fertilization or embryo development.
In the bovine, the prolonged culture period in artificial conditions (8 days) before
transfer could increase the risk. One hypothesis is that embryo manipulation
influences the imprinting of genes essential for embryo and fetal development;
another is that some inherited cytoplasmic factors that interact with the nucleus
can be modified or removed by NT techniques or during cellular fragmentation
occurring during embryo culture (for review, see Walker et al, 1996). In the
bovine, it has recently been shown that the CX43 gene encoding for gap junction
formation is expressed differently in in-vivo and in-vitro bovine embryos
(Wrenzycki et al, 1996). Such findings could help to establish a direct link
between genomic expression and early embryo morphology.
Maternal/embryonic signalling
Preimplantation embryos are known to secrete growth factors and cytokines that
could interact with their maternal environment (Schultz and Heyner, 1993; Kane
et al, 1997). Maternal/embryonic dialogue begins later for transferred embryos
and the hormonal status of the recipient and the stage of the embryo may not be
synchronous. These sub-optimal conditions could contribute to the anomalies
observed. Experiments performed on sheep on the influence of progesterone
concentrations during early pregnancy on fetal or newborn size and weight
support this hypothesis (Kleemann et al, 1994). However, if this were the case,
one would expect to observe large calves following the transfer of in-vivoderived embryos. In fact, some increase in gestation length (Kruip and den Daas,
1997) and birth weight (Wilson et al, 1995) have been reported following
MOET. It should be born in mind however, that the timing of development of
embryos in vitro differs from that in vivo and that correct synchronization is
therefore more difficult to achieve with IVP embryos. There remains therefore
the possibility that asynchronous transfers could still be involved in the large
calf syndrome.
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HJXeese, I.Donnay and J.G.Thompson
Cellular stress: a unifying mechanism to explain anomalies in early
embryo development
This review has discussed the outcome of the in-vitro manipulations to which
early embryos are subjected and some possible reasons for impaired development.
What is lacking in this field, is a framework under which the various responses
can be considered. We propose such a framework may be derived by considering
the reactions of somatic cells to stress where, in contrast with early embryos,
there is a very sizeable literature. Stress responses may be grouped into one of
four categories.
Stress proteins
Although originally identified in Drosophila melanogaster exposed to elevated
temperatures, and thus termed heat shock proteins (HSPs), the term 'stress
proteins' is increasingly being used in recognition of the variety of stimuli that
can evoke their expression (Tavaria et al, 1996). The stress proteins, which are
highly conserved, are often divided into two groups; the traditional HSPs of
which there are a number of types, and even more families (Tavaria et al, 1996)
and the glucose-regulated proteins (GRPs) (Welch, 1992), first identified as being
expressed in response to cellular glucose starvation, in turn related to a failure
of calcium homeostastis (Lee, 1987). Other stresses that induce GRPs are anoxia
and low pH. In some cells, HSPs and GRPs are regulated together (Welch, 1992)
and the glucose transporter, GLUT 1, found in most cells which consume glucose,
belongs to the GRP family (Wertheimer). The function of HSPs is unclear. They
are known to assist in the correct folding of newly-synthesized proteins (i.e. act
as molecular chaperones). It is also significant that they are induced in cells
treated with the calcium ionophore, A23187 (Welch, 1992)
Oxidative stress
Free radicals are made constantly in vivo; they are an inevitable consequence of
oxidative metabolism, and have the potential to damage cells. Anti-oxidant
defences have therefore evolved to protect cells but are not 100% efficient and
increased free radical formation is likely to increase damage; a phenomenon
known as oxidative stress. There is currently intense interest in the possibility
that free radical processes may be involved in the aetiology of many degenerative
diseases and that anti-oxidants may be able to delay or prevent their onset. Since
these facts are generally well known, the focus will be on possible embryorelated effects of free radicals, which are discussed later in this review.
Glucose metabolism
Increased glucose consumption and metabolism to lactate, is a well-known
feature of somatic cells under stress (Dominguez et al, 1996; Sviderskaya et al,
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1996) and during malignant transformation (White and McCubrey, 1995). There
are a number of possible explanations for the increase in glycolysis. Firstly, it
may compensate for a defect in oxidative metabolism and enable cells to generate
sufficient ATP, albeit less efficiently, to maintain key cellular activities such as
ion pumping, notably by the Na + , K + ATPase, which, in somatic cells, can
account for, 19-28% of ATP utilization (Rolfe and Brown, 1997). Secondly, a
switch to non-oxidative metabolism may protect cells against possible free radical
damage, as discussed above. Thirdly, a metabolism based on glycolysis will have
the effect of sparing endogenous fuels; particularly, lipid and protein, both of
which can only be utilized aerobically (Guppy et al, 1993).
The best-characterized response in glucose metabolism is an increase in deno vo synthesis (Dominguez et al, 1996) or recruitment to the plasma membrane
(Sviderskaya et al, 1996) of the ubiquitous glucose transporter, GLUT1. This
will have the effect of increasing glucose entry into the cell, where, if oxidative
metabolism is compromised, glucose conversion to lactate will be promoted, the
so-called Pasteur effect. Increased glucose entry will also serve to inhibit
respiration - the traditional Crabtree effect. It is also notable that the increase in
hexokinase activity with the development of the blastocyst, which is a feature
of in-vivo-derived embryos (Hooper and Leese, 1989), is delayed in vitro (Ayabe
et al, 1994).
Adenine nucleotides
Interference with oxidative metabolism will lead to a drop in intracellular ATP
concentration, but more especially, a dramatic rise in the concentration of AMP
(Corton et al, 1994). Not surprisingly, heat shock leads to a fall in ATP and a
rise in AMP in somatic cells, and to the activation of an AMP-activated protein
kinase cascade system which, in turn, switches off non-essential biosynthetic
pathways and spares ATP for essential activities such as the ion pumping
mentioned above.
Cellular stress in early embryos
It could be argued that the analogy between adult cells under stress and embryos
in culture is a false one; that embryos, being largely undifferentiated are somehow
'primitive' and protected against environmental insult in the female tract. One
of us has argued that early embryos show adaptation, not unlike somatic cells
(Leese, 1995) and that 'stress' can take a number of forms in vivo; for example,
there can be asynchrony between embryonic and maternal events due to delayed
fertilization or premature or delayed entry into the uterus. Moreover, the female
tract can be thought of as an immunologically hostile environment.
Stress proteins in early embryos
There are a number of indications that the 'somatic' response to heat stress is
present in early bovine embryos. For example, Hendrey and Kola (1991) found
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H.J.Leese, I.Donnay and J.G.Thompson
that microinjection of HSP70 mRNA conferred protection on mouse oocytes
exposed to a temperature of 42^43°C. Edwards and Hansen (1996) reported that
exposure of 2-cell bovine embryos to an elevated temperature (42 versus 39°C)
induced the premature synthesis of HSP68, which is not normally expressed until
genome activation at the 8-16-cell stage. Christians et al. (1995) found that
HSP70.1, the earliest gene to be expressed at the onset of zygotic gene activation
in the mouse, is then repressed before the completion of the second round of
DNA replication. However, this repression is associated with in-vitro culture and
possibly, with oxidative stress, since there was a drop in expression in the
presence of the antioxidant, Cu-Zn superoxide dismutase.
Expression of inducible (i.e. heat-induced in somatic cells) HSPs begins at the
morula stage (Ho et al, 1994; Christians et al, 1997). It is notable that HSP70
expression is 5-15-fold higher in cultured embryos (Christians et al, 1995). As
the same group comment in a recent review:
'It is striking that hsp70 transcription increases with in vitro culture conditions
whereas expression of several other genes decreases. Such cultured embryos
have to cope with handling, variations of temperature and a completely different
cellular environment. All these parameters might be considered as a source of
stress . . .' (Christians et al., 1997).
Oxidative stress in early embryos
There are several lines of evidence to suggest that culture-induced blocks to
embryo development involve the formation of (and lack of cellular defence from)
free radicals, in particular, those involving reactive oxygen species (ROS) (Rieger,
1992; Johnson and Nasr-Esfahani, 1994). Most of this work has been performed
with mouse embryos, where it has been demonstrated that the addition of a
variety of radical scavengers or ROS-destroying enzymes allows development to
occur. Furthermore, intracellular values of H2O2 increase following in-vitro
culture (Johnson and Nasr-Esfahini, 1994). In the domestic species, much of the
evidence on possible effects of oxidative stress is inconclusive, with the exception
that oxygen concentration in the culture environment is known to influence
development. The optimal concentration of oxygen for the development of sheep
and cattle embryos is ~7% (Thompson et al, 1990). Furthermore, one beneficial
effect of somatic cells in co-culture has been attributed to a reduction in O2
tension in the culture environment (Watson et al, 1994); whether this effect is
mediated through free-radical production, or through another metabolic mechanism, remains to be elucidated.
Glucose metabolism in early embryos
Mouse embryos
Differences in the metabolism of embryos in vivo and in vitro have been discussed
by Leese (1991). There is strong evidence that late preimplantation mouse
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embryos increase their glycolytic metabolism in response to the stress of culture.
Gardner and Leese (1990) found that mouse blastocysts, freshly flushed from
the female tract on day 4 produced lactate at a rate of 3.6 pmol/embryo/h
(accounting for 44% of glucose consumed), whereas blastocysts generated from
day 3 flushings and cultured overnight, gave rates of lactate production of 7.9
and 5.9 pmol/embryo/h in Ml6 and mouse tubal fluid (MTF) media, (accounting
for 91 and 73% of glucose consumption) respectively. Glycogen metabolism also
differs in mouse blastocysts in utero and in vitro; there is degradation of glycogen
in the former, but not the latter, environment (Edirisinghe et al, 1984).
Mouse blastocysts which show the highest glycolytic rate are less likely to
implant following transfer (Lane and Gardner, 1996). The best mouse embryos
are those in which glucose consumption is high and lactate production low. This
observation obviously leads to questions of cause and effect. Our working
hypothesis is that when embryos are removed from the female tract, they
experience stress in a manner similar to somatic cells. Those that survive this
insult are the ones which have consumed the most glucose, but converted only
a small proportion to lactate, at least at the blastocyst stage. Some embryos
become more glycolytic because, for reasons unknown, the stress on them has
been greater. They struggle to generate sufficient ATP via this pathway; some
give rise to pregnancies, while the majority do not.
Sheep embryos
Metabolically, there is less glycolysis in embryos derived in vivo or cultured in
SOFaaBSA than in those cultured in SOF + HS, especially when the data are
expressed per cell (Gardner et al, 1994). This observation may be related to an
increased incidence of mitochondrial degeneration observed in embryos cultured
in SOF + HS than in serum-free conditions (Dorland et al, 1994).
Bovine embryos
IVP bovine blastocysts show a higher and earlier rate of aerobic glycolysis,
compared with that of their in-vivo counterparts. The maximum rate of glucose
oxidation is achieved at the morula stage by in-vitro embryos, whereas the
maximum for in-vivo embryos is at the blastocyst stage (Khurana, 1992).
A framework for research on the origins of fetal oversize
In light of the foregoing discussion on the embryonic and fetal abnormalities in
the sheep and the cow, and the effects of cellular stress on somatic and embryonic
cells, we have devised a model (Figure 1) to account for the origins of fetal
oversize in domestic animals. The model provides a framework for research at
the level of biochemistry and molecular cell biology, which would examine the coordinated expression in early embryos, removed from their natural environment, of
the following: stress proteins; oxidative stress and mitochondrial function; AMP
kinases; GLUT transporters; and metabolic enzymes
195
HJ.Leese, I.Donnay and J.G.Thompson
Embryo manipulation
and conditions of culture
Apoptosis
Embryo fragmentation
Modified
genomic expression
Abnormal compaction
Modified kinetics
Modified ICM/TE
allocation
Maternal/embryo
asynchrony
Modified
foetal/placental ratio
Modified
foetal/placental metabolism
Figure 1. Model for origin of large calf/lamb syndrome.
The importance of this research is that it would provide markers of the extent
of cellular injury, data on which, for early embryos are woefully inadequate.
Thus, when the majority of studies on preimplantation embryos seem content to
measure blastocyst formation as sole endpoint, it is not surprising that we
understand so little about the aberrant physiology which underlies fetal oversize.
The focus needs to shift to the question of what, in physiological, biochemical
and molecular terms, makes a viable blastocyst.
The early origins of adult disorders in man
No review on the embryo and fetal origins of later disorders is complete without
mention of the work of the UK epidemiologist, David Barker. Based on an
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Assisted conception
exhaustive analysis of 80 year old medical records, Barker discovered striking
associations between anthropometric measures such as birth weight and weight
at 1 year and the susceptibility to certain disorders in the adult, including coronary
heart disease, stroke, hypertension and non-insulin dependent diabetes (Barker,
1993). For example, death from ischaemic heart disease in men increases as
birth weight or weight at 1 year decreases. The situation becomes more complex
since different risk factors are associated with different patterns of early growth;
adult blood pressure is inversely related to birth weight, but not independently
to weight at 1 year - and the strongest predictor of adult blood pressure is, in
fact, a large placenta in relation to birth weight.
Hales and Barker (1992) have put forward the concept of 'metabolic setting'
to explain these associations, particularly in relation to the onset of non-insulin
dependent diabetes. In essence, they propose that poor maternal and early
postnatal nutrition lead the conceptus to adopt a 'thrifty metabolism' in an
attempt to adapt to the diminished supply of nutrients and that this 'programming'
persists throughout life. Problems begin when the offspring is confronted with a
normal, as opposed to marginal diet, or one which is nutrient rich; its metabolism
is wrongly programmed and metabolic defects begin to set in. For a full account
of this hypothesis, see the excellent review by Desai and Hales (1997). In the
context of the embryo - if conditions in utero or in vitro are abnormal, and in
particular, if the diet is too 'rich' - then metabolic resetting will take place which
will affect the subsequent fetal growth and development and lead to the problems
of oversize discussed in this review. These ideas are gaining rapidly in credence
and are likely to become centre-stage and remain so for some time to come.
Economic and technological factors in the origins of fetal oversize
The large calf/lamb/fetal abnormality problems can be seen as a consequence of
advances driven by biotechnological goals rather than arising from basic scientific
research. Thus, although multiple forces determine the direction of science, they
may be reduced, in the simplest model, to two: (i) the 'push' that comes from
the disinterested pursuit of knowledge for its own sake; traditional basic research;
and (ii) the 'pull' that comes from the needs of commerce.
Push
—> Scientific Progress
—> Pull
While advances in assisted conception technologies originally contained a
substantial element of 'push' in the form of detailed information on early embryo
development, mainly derived from work on rodents, recent developments have
been 'pulled' to a large extent by the needs of infertile couples and for advances
in farm animal production, both of which have spawned substantial industries.
Worldwide, the pendulum has shifted to advances driven more by technology
than by basic science. As a result, the undoubted benefits that will derive from
applying the new genetics to agriculture, have brought with them undesirable
side effects. The agricultural industries would obviously like quick solutions to
197
HJ.Leese, I.Donnay and J.G.Thompson
these problems but it is our contention that these will come slowly and only
after the belated allocation of resources into understanding the physiology of
early mammalian development. As Einstein once put it: 'Science will stagnate
if it is made to serve practical goals'.
Lessons for human assisted conception
In the human, conventional IVF techniques do not result in an increase in the
birth of abnormal babies. However, in the light of the observations made
in livestock species, the recent introduction of new technologies such as
intracytoplasmic sperm injection (ICSI), embryo biopsy, cytoplasmic transfer, invitro maturation and prolonged embryo culture in humans need to be monitored
with extreme care, as exemplified by the work of Bonduelle et al. (1996) and
allied with a long-term programme of research on the responses of early embryos
to stress. Similar concerns about the potential health problems arising out of
human assisted conception programmes have been raised by Seamark and
Robinson (1995).
Acknowledgements
I.D. and H.J.L. acknowledge financial support from the European Commission during the writing
of this review.
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