Department of Obstetrics and Gynaecology, University of Adelaide, SA 5005, Australia
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Abstract |
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Key words: embryo culture/epigenetic/fetal development/gene expression/placenta
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Introduction |
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Several articles published over recent years warn of the implication of ART techniques on subsequent fetal growth, birth weight and adult health (Seamark and Robinson, 1995; Leese et al., 1998
; Boerjan et al., 2000
; Khosla et al., 2001
). All call for long-term analysis of children conceived by ART, primarily because of the epidemiological link between small for gestational age babies and adult onset diseases, such as cardiovascular disease and type II diabetes [the fetal origins of adult disease hypothesis (Barker, 1998
)] Despite this, comparatively few studies have examined neonatal and longer-term effects, even in animal models. Why? There is no doubt that such studies are expensive and logistically difficult. Patient identity and confidentiality also pose significant ethical challenges. However, an additional reason may be lack of commitment to provide an evidence-based analysis. This position is becoming increasingly untenable.
We would like to further broaden the debate by drawing attention to the likely consequences for fetal development of changes in gene expression brought about by a less than optimal physiochemical environment early in life. We acknowledge that for some technologies, such as ICSI of immature sperm and nuclear transfer, direct epigenetic alteration of gene expression is the most plausible origin of subsequent abnormality. Such manipulations alter methylation and histone acetylation patterns to reprogram nuclear structure (Renard et al., 2002). Moreover, clear causal pathways (Figure 1
) link epigenetically altered gene expression patterns, especially of imprinted genes, with determinants of fetal growth, such as placental development and fetal/placental metabolism (Young, 2001
). De Rycke and colleagues cite extracellular environment during critical periods of development as a second key mechanism underlying epigenetic modification (De Rycke et al., 2002
). As previously noted (Leese et al., 1998
), in-vitro culture and manipulation alter oocyte and embryo cell physiology by stress-induced cellular responses, which in turn alter early gene expression patterns. This can be achieved either by epigenetic mechanisms, such as a change in methylation status during global remethylation early in preimplantation development, or by environmentally-mediated effects on transcriptional regulation. Thus, influences on gene expression may not be the direct result of culture conditions or physical manipulation, but elicited by mechanisms invoked by stress pathways (such as cellular apoptosis or compromised metabolic state). Here we propose a causal model which, if proven, would explain how a range of perturbed extracellular environments and embryonic manipulations can each lead to altered phenotypes in offspring. We also discuss the known mediators of environmentally-signalled responses and examine the relationship between in-vivo environmental manipulation (mediated via dietary manipulation) and ex-vivo environments encountered during in-vitro culture. In addition, we ask whether alterations in fetal development resulting from ART could lead to an increased risk of adult disease, as a shift to lighter birth weights, independent of the effects of multiple births, is now recognized as a consequence of IVF technology in humans (Koudstaal et al., 2000
; De Rycke et al., 2002
).
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A stress-induced, causal model |
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Environmental stressors potentially influencing early embryo programming |
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Are there credible associations between dietary-induced in-vivo environments and in-vitro culture conditions? Low protein diets during early pregnancy influence maternal factors, for example decreasing plasma insulin, increasing glucose and decreasing the concentration of a number of essential amino acids (Kwong et al., 2000). In addition, high protein diets in ruminants during the peri-conceptual period have been associated with altered intrauterine pH (Butler, 1998
) and high plasma ammonia levels (McEvoy et al., 1997
, 2001
). Such factors during ex-vivo culture are known to negatively influence the kinetics and quality of embryo development. Ammonia itself is arguably the best characterized facilitator of large offspring syndrome in ruminants (McEvoy et al., 1995
) and micromolar levels during mouse embryo culture cause significant fetal growth retardation and exencephaly following transfer (Lane and Gardner, 1994
). Importantly, different preparations and commercial batches of culture media are likely to vary in their concentrations of ammonia, as it is a common contaminant of amino acid preparations, albumins and sera used to supplement media. Protein-free preparations are not immune to possible ammonia-related effects, as there is evidence that intracellular ammonia production is higher in embryos cultured under protein-free conditions compared with protein-supplemented conditions (Thompson, 2000
). This may partly explain observations that removal of protein from embryo culture systems does not equate to absolution of perturbed phenotypes (Kaye and Gardner, 1999
; Hartwich et al., 2000
).
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Epigenetics and placental function |
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In-vitro culture in ruminants is linked with defective placentation (Thompson and Peterson, 2000; Bertolini and Anderson, 2002
). We have shown that in cows, in-vitro embryo production can yield fetuses with abnormal allantoic development and thus failed angiogenesis of placentation at day 35 of pregnancy (Thompson and Peterson, 2000
). Failure of placental vascular development at day 35 of pregnancy has also been reported in placentas from cloned fetal sheep (De Sousa et al., 2001
). In contrast, Bertolini and Anderson have recently described in-vitro embryo culture leading to oversized bovine fetuses and to a few overly large placentomes (Bertolini and Anderson, 2002
). Placentomegaly with a greatly expanded junctional zone but fetal growth restriction is common in the few surviving mouse clones (Tanaka et al., 2001
). Surprisingly, there appears to be little information on the placental structure following culture and transfer of mouse embryos. However, it has recently been shown that fetuses on day 18 of gestation from in-vitro cultured and transferred mouse embryos had reduced fetal weights, similar placental weights, but a lower fetal:placental weight ratio than fetuses derived from the transfer of in-vivo-derived (control) embryos (Sjöblom et al., 2001
). Furthermore, significant differences at the histological level were also observed, with cultured embryos exhibiting increased junctional zone and reduced labyrinthine areas (Sjöblom et al., 2001
).
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Conclusions |
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Together, these considerations underscore the necessity for sustained, long-term tracking of the health of children and adults conceived through ART practices. These studies are of paramount importance, despite the difficulties involveduntil their completion, human ART procedures remain a series of experiments in-progress.
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Acknowledgements |
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Notes |
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References |
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