According to the foetal programming hypothesis, factors influencing growth and development in the prenatal and early postnatal periods may affect vulnerability to chronic diseases (e.g., type 2 diabetes, coronary heart disease, etc.). Animal studies suggest that the neuroendocrine system, in particular the hypothalamic pituitary adrenal (HPA) axis, is the primary biological pathway through which this occurs. This has prompted interest in the factors that may affect the foetal HPA axis in humans. Several plausible candidates exist including: prenatal maternal factors that may affect the intra-uterine environment (e.g., depression, smoking etc.) and genes affecting foetal development by regulating the maternal axis (e.g., glucocorticoid receptor and 11beta-hydroxysteroid dehydrogenase (11beta-HSD) isoforms1 and 2 genes). However, the human evidence regarding foetal programming of the HPA axis remains equivocal. This is due, in part, to (i) limitations of existing approaches to assessing the human neuroendocrine system and (ii) a paucity of relevant data on potential environmental determinants of prenatal maternal HPA activity.

This study has sought to overcome these limitations by undertaking a more rigorous assessment of the neuroendocrine system than has previously been possible, through the implementation of the CO2 test. This test provides measures of HPA, sympathetic and parasympathetic reactivity and recovery to a stressor. Furthermore, as our enquiry is based in the ALSPAC (Avon Longitudinal Study of Pregnancy & Childhood) cohort in which environmental data are available on mothers and their offspring, we will be able to examine the relationship between foetal and early life factors (in both mother and offspring) & neuroendocrine activity in offspring at 16 years.

Data we have collected/already have approval to access (ALSPAC proj. B277):

An acute novel stressor (the 'CO2 test') was used in a sub-sample of ALSPAC (N=142). The test involved a single inhalation of 65% oxygen/35% CO2. This acts as a transient physiological challenge activating the key stress-responsive neuroendocrine systems. Consequently, the task produces increases in cortisol, blood pressure and plasma noradrenaline concentrations and an immediate reduction in heart rate that precedes the rise in blood pressure.

In order to explore inter-individual differences in sympathetic and parasympathetic reactivity and recovery, blood pressure and heart rate were recorded every minute from 5 minutes prior to the inhalation and for the 5 minutes post inhalation. Heart rate variability was recorded by the use of a Polar watch over this same time period as the R-R intervals provide assessments of the dynamic vagal regulation of the heart and dynamic changes in baroreceptor sensitivity. HPA activity was captured by the measurement of salivary cortisol (using salivettes) immediately pre-inhalation (-1 minute) and 10, 20, 30 minutes post-inhalation.

In order to control for factors known to affect salivary cortisol: all analyses will allow for time of day when samples were collected and stage in menstrual cycle will be considered for inclusion in the analysis. Participants were also asked to refrain from food and caffeine for at least 1 hour prior to the CO2 task until the final saliva samples have been taken.

In addition 2 psychological measures were administered prior to the CO2 test, the profile of mood states for adolescents (POMS-A) and the 10 item perceived stress scale (PSS-10). This was necessary to allow for inter-individual differences in mood on the day of the stress test.

Existing data on mothers and children will be collated as previously agreed. For mothers, this will focus on prenatal self-reports of social and emotional exposures captured through: (i) indices of quality of social environment; (ii) social support; (iii) distress and (iv) stressful life events. Data on health behaviour exposures (i.e., diet, smoking and alcohol consumption) will also be collated to examine the extent to which the effects of social and emotional exposures on offspring neuroendocrine activity are mediated by health behaviours.

For children, information will be extracted on birth weight, ponderal index and birth weight corrected for gestational age. While the effects of puberty on the neuroendocrine system are not expected to be significant, we will collate growth information from birth and, for girls, data on start of menses so that the effects of pubertal development on neuroendocrine outcomes can be examined.

What biological samples we would like access to and why:

We are seeking to extend the scope of our original application by requesting access to the fasting bloods samples for the 142 volunteers who participated in the stress test at the most recent recall of the cohort (2007/08). We propose to assay plasma cortisol from these samples in order to address the following issues:

1. Explore the relationship betweeen a measure of basal HPA axis function (i.e., plasma cortisol) and measures of HPA reactivity and recovery (i.e., salivary cortisol responses to the CO2 stress test).

2. Examine the relationship between foetal and early life factors and plasma cortisol and whether and how this relationship differs from relationships observed with salivary cortisol.

In addressing these issues, we will also be able to explore the 'added value' conferred by 'stress testing' in this cohort and the most appropriate primary outcomes for future research.

Thus, we are requesting access to 25 ul of the plasma samples collected in heparin containers, from the 142 participants who have participated in our preliminary study. These assays would be conducted in Clinical Biochemistry at the University of Bristol under the supervision of Professor Stafford Lightman.