Exposing the link between placental endocrine dysfunction and offspring behavioural outcomes

There is a well-defined association between early life adversity (either prenatally or in early childhood) and significantly poorer outcomes for children. Exposure of the developing fetus, for example to poor diet, and/or exposure of very young children to suboptimal maternal care has life long consequences including the increased occurrence of ADHD, depression and schizophrenia. However, maternal mood disorders during pregnancy and in the immediate postnatal period are also intimately linked to poor diet in pregnancy. Dissecting apart the relative contributions of these exposures is challenging in human cohort studies.

We have developed a novel animal model, based on genetic modification of an imprinted gene called Phlda2, which models an alteration observed in the placenta after female mice are fed a low protein or a high fat diet during pregnancy also seen in human placenta from women reporting poor diet choices in pregnancy. Using our animal model, we have shown that elevated placental Phlda2 results in dysfunctional placental development and low birth weight. A key change we observe in the placenta is a reduction in the number of cells manufacturing placental hormones. These hormones are important for both fetal growth and maternal care. We have found that the female mice exposed to the mutant placenta neglect their pups. In humans, both low birth weight and poor maternal care have been linked to problems later in life including the increased prevalence of mental health issues. We have recently performed pilot work to examine offspring behaviour in our model. Remarkably, we found that both the offspring carrying the transgene and their littermates behaved abnormally showing signs of depression. This tells us that it is not the gene change that causes the abnormal behaviour. Instead there must be something about the shared environment which causes these changes in behaviour. This could be the exposure in utero to low placental hormone levels, poor fetal growth or poor quality maternal care after birth or potentially the combined adversities. We are able to dissect apart the contribution of the in utero environment versus postnatal exposures using a simple cross fostering techniques, experiments that are planned in this proposal. However, we wish to take this investigation even further. We know that maternal diet during pregnancy alters the expression of our gene in the placenta. Suboptimal maternal diets have been linked in humans and experimental models to both poor maternal care and abnormal offspring behaviour. This raises the intriguing possibility that placental endocrine dysfunction induced by a poor maternal diet contributes to both the abnormal behaviour of the mother and her child via placental dysfunction. We will combine our genetic model with a dietary model to examine this relationship in detail. This work is exceptionally timely as we are leading the studies on placental endocrine dysfunction using this model. We are in a unique position to build on strong preliminary data from our groups underpinning each aspect of the planned work, and we have the relevant expertise to ensure we achieve our aims.

In addition to examining this novel mechanism linking early life adversity to later life outcomes, our work may have translational relevance. We know that 25% of human babies who are low birth weight due to fetal growth restriction have elevated placental PHLDA2. We have recent data linking elevated placental PHLDA2 to altered behaviour in human infants. We can ensure the maximum impact of our animal studies by translating directly to human pregnancy via our Grown in Wales study funded by the MRC. Consequently, our remarkable findings on maternal lifestyles during pregnancy funded by the BBSRC could rapidly lead to better outcomes for human mothers and their children.

We propose that placental endocrine dysfunction induced by aberrant expression of the imprinted gene Phlda2 links adverse maternal diets to later life behavioural outcomes in offspring. We have shown that placental expression of Phlda2 is elevated in response to adverse maternal diets (low protein, high fat). These diets have been linked to both poor maternal care and abnormal offspring behaviour. In a mouse model (2XPhlda2), we find significant defects in a key endocrine lineage of the mouse placenta concurrent with low birth weight. Wild type dams exposed to 2XPhlda2 mutant placenta neglect their pups. Remarkably, both Phlda2 transgenic mice and their non-transgenic littermates display abnormal hedonistic behaviours. The presence of abnormal behaviour in non-transgenic offspring indicates a non-genetic mechanism.

To dissect apart the complex prenatal and postnatal relationships, we will use a simple cross fostering experiment where in utero exposed pups will be raised by control dams and control pups raised by dams exposed to mutant placenta followed by a behavioural and molecular characterisation to quantify key changes, and the degree of attenuation by cross fostering. We will ask whether normalising placental endocrine signaling in utero restores offspring behaviour and then assess the relevance of fetal growth restriction. Finally, we will be perform a key experiment combining a genetic model in which elevation of placental Phlda2 is prevented with a low protein dietary model to determine the contribution of elevated placental Phlda2 to low protein diet-induced changes in offspring behaviour that have been widely reported. All of the work proposed will use existing models and expertise with Home Office approval in place for the work program. This work is fundamentally important and timely providing novel insight on a mechanism contributing to life long behaviour, which may have translational relevance in humans.

Enhancing quality of life, health and well-being:
While are studies are in rodents, which have very different placenta to humans, they may have some relevance to our understanding of human mental health disorders. We have some evidence from our MRC funded study that altered expression of PHLDA2 in the placenta is associated with increased infant aggression. We also have evidence that maternal diet during a human pregnancy elevates placental PHLDA2. These studies are in the earliest stages but this proposed BBSRC study will obtain evidence to support a causal relationship between placental dysfunction and offspring behavioural outcomes. Moreover, this work in rodents could lead to a better understanding of how maternal diet contributes to the healthy development of their children to enhance the quality of life.

The economy:
If our studies translate to humans, our discoveries have the potential to benefit the British economy by reducing costs to the NHS in treating childhood behavioural disorders. Suboptimal maternal diets can cause fetal growth restriction and substantially poorer lifelong outcomes for offspring which place a substantial burden on the economy.

Commercialisation:
Although not the focus of this study, there is the potential for the development of biomarkers predicting offspring outcomes. This could potentially involve examining the expression of key genes in the term placenta. Ideally, we would like to identify factors present in maternal serum, which could be picked up in pregnancy using a simple blood test, which could also support preventative straggles, as well as predicting outcomes.

Contributing towards evidence based policy-making and influencing public policies and legislation at a local, regional, national and international level:
Phlda2 is elevated in the placenta in response to a suboptimal maternal diet in rodents. We have found similar increases in expression of PHLDA2 in human placenta obtained from women reporting unhealthy lifestyles during pregnancy. Our current work suggests that elevated placental Phlda2 is relevant to both fetal and maternal health, and long life mental health. Our work could lead to changes in the Public Health policies concerning advice to and management of pregnant women.

Contributing to increasing public awareness and understanding of science, economic and societal issues:
The public will be very interested in our work as it concerns healthy lifestyles in pregnancy and the wellbeing of children. Our work will raise awareness of the important of health lifestyles in pregnancy in shaping later life outcomes for children

Enhancing the knowledge economy:
Nothing is published linking Phlda2 to childhood outcomes. Our scientific discoveries will provide new knowledge.

Worldwide scientific advancement to address issues of importance in other countries or globally: Our work may be even more relevant to populations where dietary-induced low birth weight is more prevalence such as developing countries where the occurrence of low birth weight is two-fold higher than in the UK.

Delivering and training highly skilled researchers:
This work will provide valuable training to a young researcher in in vivo (reproduction, behavioural neuroscience), molecular (RNA-seq, IHC) and big data skills (Bioinformatics). The technician's skill set will be enhanced by exposure to laboratory techniques, animal husbandry, surgery (recipient transfer) and cross fostering.