Epigenetic risks related to assisted reproductive technologies

Short- and long-term consequences for the health of children conceived through assisted reproduction technology: more reason for caution?

Jeremy G. Thompson1, Karen L. Kind, Claire T. Roberts, Sarah A. Robertson and Jeffrey S. Robinson

Department of Obstetrics and Gynaecology, University of Adelaide, SA 5005, Australia


    Abstract
 Top
 Abstract
 Introduction
 A stress-induced, causal model
 Environmental stressors...
 Epigenetics and placental...
 Conclusions
 Acknowledgements
 References
 
Does the manipulation of gametes and embryos as practised in human IVF invoke perturbations in fetal and neonatal phenotype? There is increasing evidence that the answer is ‘yes’, although the degree of perturbation may be less acute than observed in other species. However, the long-term consequences are not known, and may prove to be considerable. There is now a substantial body of evidence from animal models suggesting that assisted reproductive technologies (ART) are associated with altered outcomes in fetal and neonatal development. Epigenetic modification of gene expression is an attractive hypothesis that accounts for these differences and is one of a number of causal pathways that may be activated by cellular stress invoked during manipulation. Here we widen the debate to propose that environment-induced cellular stress also acts to modify fetal and placental gene expression, potentially also contributing to phenotype skewing after ART.

Key words: embryo culture/epigenetic/fetal development/gene expression/placenta


    Introduction
 Top
 Abstract
 Introduction
 A stress-induced, causal model
 Environmental stressors...
 Epigenetics and placental...
 Conclusions
 Acknowledgements
 References
 
The influence of ex-vivo conception and embryogenesis on the health of the ensuing fetus and neonate remains an intriguing and topical question. The review article by De Rycke and colleagues (De Rycke et al., 2002Go) is an excellent addition to this debate, further highlighting the potential health dangers for children born from assisted reproductive technologies (ART). Their analysis sounds a clear warning that epigenetic modification to gene expression occurs in several species as a consequence of ART techniques. Their evidence that in-vitro culture not only alters the expression of genes thought to be central to embryo development (Niemann and Wrenzycki, 2000Go), but specifically affects the methylation status of parentally imprinted genes (Khosla et al., 2001Go; Young et al., 2001Go), should arouse the concerns of all who work in human ART.

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, 1995Go; Leese et al., 1998Go; Boerjan et al., 2000Go; Khosla et al., 2001Go). 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, 1998Go)] 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., 2002Go). Moreover, clear causal pathways (Figure 1Go) link epigenetically altered gene expression patterns, especially of imprinted genes, with determinants of fetal growth, such as placental development and fetal/placental metabolism (Young, 2001Go). De Rycke and colleagues cite extracellular environment during critical periods of development as a second key mechanism underlying epigenetic modification (De Rycke et al., 2002Go). As previously noted (Leese et al., 1998Go), 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., 2000Go; De Rycke et al., 2002Go).



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Figure 1. A stress-induced, causal model. Manipulation of gametes and embryos results in cellular stress. There are several potential pathways, three of which are shown here, in which stress can modulate cellular function. The response may involve several pathways that are interactive. The degree of response will determine the degree of perturbation, which is lethal at its most calamitous. Adapted from Leese et al. (1998)Go.

 
Lastly, we consider placental gene expression, as animal studies now compellingly implicate the placenta in mediating the consequences of ART. Indeed, it has been argued that many of the fetal pathologies observed are manifestations of placental defects (Cross, 2001Go; Bertolini and Anderson, 2002Go).


    A stress-induced, causal model
 Top
 Abstract
 Introduction
 A stress-induced, causal model
 Environmental stressors...
 Epigenetics and placental...
 Conclusions
 Acknowledgements
 References
 
In-vitro manipulation, in particular ex-vivo culture, is known to alter the cellular biochemistry of early cleavage stage embryos. In particular, changes in morphology (Thompson, 1997Go), embryonic blastomere survival (Hardy and Spanos, 2002Go), gene expression (Niemann and Wrenzycki, 2000Go) and metabolism (Thompson, 1997Go) have all been observed. Leese et al. proposed that in-vitro manipulation is therefore a stress-inducing phenomenon and that resulting fetal pathologies are the outcome of stress-activated pathways, the most severe of which are lethal (Figure 1Go) (Leese et al., 1998Go). We have refined the original model to include epigenetic modification amongst other stress-induced mechanisms effecting change in transcriptional regulation of genes (Figure 1Go). Intracellular parameters such as pH and REDOX state are influenced by a number of extracellular and intracellular factors and are perturbed during in-vitro culture to varying degrees. Changes in intracellular REDOX state, in particular, are known to significantly alter gene expression patterns (Harvey et al., 2002Go). The degree to which environmentally-regulated transcription factor families, such as the basic helix–loop–helix PAS-domain family, influence early embryo development has yet to be fully elucidated, but such transcription factors are already implicated in the response to chemical insults including dioxin (Tscheudschilsuren et al., 1999Go). The potential influence of survival factors (e.g. growth factors and cytokines) on programming of embryo development provides a further, as yet unexplored, avenue linking environment with gene transcription. Furthermore, whether parameters including inner cell mass (ICM):trophectoderm (TE) cell ratios and metabolic programming in early embryos can be reflected in fetal:placental tissue ratios or metabolic profiles respectively (see Figure 1Go) requires considerable research. Nevertheless, this model may assist in explaining the between-species variation in the degree of abnormalities observed in response to ART, with ruminants (especially sheep and cattle) being the most susceptible and, to date, humans being perhaps the least. Different levels of sensitivity to perturbations in each pathway may exist between species, with outcomes related to the degree of pathway activation.


    Environmental stressors potentially influencing early embryo programming
 Top
 Abstract
 Introduction
 A stress-induced, causal model
 Environmental stressors...
 Epigenetics and placental...
 Conclusions
 Acknowledgements
 References
 
Manipulation of the maternal diet, even briefly during early pregnancy, has been shown to affect subsequent embryo development and to have neonatal consequences. Diets both high and low in protein content can have detrimental effects. High protein diets in sheep during the peri-conceptual period have been linked with low embryo survival and high birth weights similar to the large offspring syndrome (McEvoy et al., 2001Go). Low protein diets during early pregnancy in rats, specifically during the period between zygote and blastocyst stages of development, were found to significantly reduce the birth weight of resulting pups. Embryos recovered at day 4 from low protein-fed rats comprised fewer inner mass cells and a lower ICM:TE cell ratio (Kwong et al., 2000Go). Precisely how dietary manipulations affect the oviductal or uterine environment to cause these effects has yet to be elucidated, and indeed may be directly linked to epigenetic changes in methylation status (Wolff et al., 1998Go). Nevertheless, experimental studies in animal models have clearly linked compromised maternal nutrition to altered prenatal growth and adverse outcomes in terms of cardiovascular and metabolic function in adult offspring (Kwong et al., 2000Go; Bertram and Hanson, 2001Go; Langley-Evans, 2001Go). Most intriguingly, similar results following embryo culture are now also being reported. An association in mice between culture and lighter fetal weight and increased post-natal adiposity, together with body weight gain and skewed organ morphometry in adults, has been identified (Sjöblom et al., 2001Go). Compared with in-vivo derived embryos, culture reduced total cell number and ICM:TE cell ratio. Interestingly, culture in the presence of granulocyte-macrophage colony stimulating factor (a growth factor known to promote embryo development in vitro) mostly overcame these embryonic and phenotypic differences (Sjöblom et al., 2001Go). These data illustrate the scope for alleviating the effects of ex-vivo culture by improving the culture environment to more closely reflect that provided in vivo.

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., 2000Go). In addition, high protein diets in ruminants during the peri-conceptual period have been associated with altered intrauterine pH (Butler, 1998Go) and high plasma ammonia levels (McEvoy et al., 1997Go, 2001Go). 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., 1995Go) and micromolar levels during mouse embryo culture cause significant fetal growth retardation and exencephaly following transfer (Lane and Gardner, 1994Go). 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, 2000Go). This may partly explain observations that removal of protein from embryo culture systems does not equate to absolution of perturbed phenotypes (Kaye and Gardner, 1999Go; Hartwich et al., 2000Go).


    Epigenetics and placental function
 Top
 Abstract
 Introduction
 A stress-induced, causal model
 Environmental stressors...
 Epigenetics and placental...
 Conclusions
 Acknowledgements
 References
 
Perturbations of imprinting in the placenta that affect placental function may have indirect effects on the fetus, irrespective of whether imprinting is altered in the fetus itself. A cluster of imprinted genes found mostly on mouse chromosome 7 and human chromosome 11 (Table IGo) have been shown by gene deletion and experimental uniparental disomy studies to play a role in placental differentiation and function. Thus, it is unlikely that dysregulation of any single imprinted gene is responsible for subsequent effects on placental function resulting from a suboptimal pre-implantation environment. Indeed it has been shown that some of these genes regulate the expression of others in this cluster (Caspary et al., 1998Go, 1999Go; Grandjean et al., 2000Go).


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Table I. Imprinted genes regulating placental differentiation and function
 
It has been suggested that defective imprinting in the placenta plays a role in the aetiology of pre-eclampsia, a common hypertensive disorder of pregnancy which is associated with poor extravillous cytotrophoblast invasion of the decidua, intrauterine growth restriction of the fetus and can result in maternal death (Dekker and Sibai, 1998Go; Graves, 1998Go). While a specific culprit gene has not been identified, it has been argued (Graves, 1998Go) that a paternally imprinted (maternally active) gene might be to blame. Although tantalising, the hypothesis does not easily explain the fact that pre-eclampsia is generally a disease of first pregnancy (Dekker and Sibai, 1998Go). However, it is quite conceivable that faulty imprinting plays a role in the disease.

In-vitro culture in ruminants is linked with defective placentation (Thompson and Peterson, 2000Go; Bertolini and Anderson, 2002Go). 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, 2000Go). Failure of placental vascular development at day 35 of pregnancy has also been reported in placentas from cloned fetal sheep (De Sousa et al., 2001Go). 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, 2002Go). Placentomegaly with a greatly expanded junctional zone but fetal growth restriction is common in the few surviving mouse clones (Tanaka et al., 2001Go). 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., 2001Go). Furthermore, significant differences at the histological level were also observed, with cultured embryos exhibiting increased junctional zone and reduced labyrinthine areas (Sjöblom et al., 2001Go).


    Conclusions
 Top
 Abstract
 Introduction
 A stress-induced, causal model
 Environmental stressors...
 Epigenetics and placental...
 Conclusions
 Acknowledgements
 References
 
There is considerable evidence demonstrating that ART techniques applied to gametes and embryos can cause epigenetic changes leading to altered fetal development, some of which will be elicited via direct effects on chromatin structure and remodelling. However, we conclude that epigenetic modification is best viewed as a component of a broader causal model linking environmental stressors with phenotype perturbation through both transcriptional and epigenetic modification of gene expression. The possibility of a more complex two-way interaction between epigenetic change and other stress-induced pathways cannot be excluded—it is a hypothesis that requires considerable research.

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 involved—until their completion, human ART procedures remain a series of experiments in-progress.


    Acknowledgements
 Top
 Abstract
 Introduction
 A stress-induced, causal model
 Environmental stressors...
 Epigenetics and placental...
 Conclusions
 Acknowledgements
 References
 
Our thanks to Dr Lisa Edwards (Department of Obstetrics and Gynaecology, University of Adelaide) for her helpful discussions and review of the manuscript.


    Notes
 
1 To whom correspondence should be addressed at: Reproductive Medicine Unit, Department of Obstetrics and Gynaecology, University of Adelaide, The Queen Elizabeth Hospital, Woodville Road, Woodville, South Australia, 5011. E-mail: jeremy.thompson{at}adelaide.edu.au Back


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 Abstract
 Introduction
 A stress-induced, causal model
 Environmental stressors...
 Epigenetics and placental...
 Conclusions
 Acknowledgements
 References
 
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