Rebecca Reynolds

Glucocorticoid excess and the developmental origins of disease: Two decades of testing the hypothesis — 2012 Curt Richter Award Winner

Low birthweight, a marker of an adverse in utero environment, is associated with cardiometabolic disease and brain disorders in adulthood. The adaptive changes made by the fetus in response to the intra-uterine environment result in permanent changes in physiology, structure and metabolism, a phenomenon termed early life programming. One of the key hypotheses to explain programming, namely over exposure of the developing fetus to glucocorticoids, was proposed nearly two decades ago, following the observation that the fetus was protected from high glucocorticoid levels in the mother by the actions of the placental barrier enzyme, 11β-hydroxysteroid dehydrogenase, which converts active glucocorticoids into inactive products. Numerous mechanistic studies in animal models have been carried out to test this hypothesis using manipulations to increase maternal glucocorticoids. Overall, these have resulted in offspring of lower birthweight, with an activated hypothalamic-pituitary-adrenal (HPA) axis and an adverse metabolic profile and behavioural phenotype in adulthood. Altered glucocorticoid activity or action is a good candidate mechanism in humans to link low birthweight with cardiometabolic and brain disorders. We have carried out detailed studies in men and women showing that high levels of endogenous glucocorticoids, or treatment with exogenous glucocorticoids, is associated with an adverse metabolic profile, increased cardiovascular disease and altered mood and cognitive decline. Our laboratory carried out the first translational studies in humans to test the glucocorticoid hypothesis, firstly demonstrating in studies of adult men and women, that low birthweight was associated with high fasting cortisol levels. We went on to dissect the mechanisms underlying the high fasting cortisol, demonstrating activation of the HPA axis, with increased cortisol responses to stimulation with exogenous adrenocorticotrophin hormone, lack of habituation to the stress of venepuncture, and increased cortisol responses to psychosocial stress. We have developed new dynamic tests to dissect the mechanisms regulating HPA axis central negative feedback sensitivity in humans, and demonstrated that this may be altered in obesity, one component of the metabolic syndrome. There are now studies in humans demonstrating that high circulating levels of maternal cortisol during pregnancy correlate negatively with birthweight, suggesting that excess glucocorticoids can by-pass the placental barrier. Deficiencies in the barrier enzyme, potentially increasing fetal glucocorticoid exposure, can also arise in association with maternal stress, malnutrition and disease, and can be inhibited by consumption of liquorice, which contains glycyrrhizin, an HSD inhibitor. Importantly, studies in humans have now demonstrated that high maternal cortisol in pregnancy and/or inhibition of HSD2 are associated with programmed outcomes in childhood including higher blood pressure, behavioural disorders as well as altered brain structure. We are investigating this further, using novel magnetic resonance imaging techniques to study the developing fetal brain in utero. The translational studies in support of the glucocorticoid hypothesis, and demonstrating that glucocorticoids are both mediators and targets of programming, are exciting and raise the question of whether this information can be used to identify those individuals most at risk of later life disease. In a recent study we showed that alterations in DNA methylation at genes important in regulating cortisol levels, tissue glucocorticoid action, blood pressure and fetal growth, are present in adulthood in association with both early life parameters and cardiometabolic risk factors. These preliminary data add to the limited literature in humans indicating a persisting epigenetic link between early life events and subsequent disease risk. Such findings open novel avenues for further exploration of the contribution of glucocorticoids to later life disease.

What is the evidence in humans that DNA methylation changes link events in utero and later life disease

Development in utero is now recognized as crucial to determining later life disease susceptibility. Whilst mechanisms are poorly understood, there has been considerable interest in the potential role of epigenetic processes in intra‐uterine programming of disease. Epigenetic modifications include various mechanisms that influence chromatin structure and gene expression. Here, we review emerging data from human studies that altered DNA methylation links intra‐uterine events with later life disease. Further research in this field is needed to determine whether altered DNA methylation in target tissues can be used as a biomarker for the early identification of and intervention in individuals most at risk of later life disease.

Maternal depressive symptoms throughout pregnancy are associated with increased placental glucocorticoid sensitivity

BACKGROUNDnMaternal prenatal depression predicts post-partum depression and increases risk of prematurity and low birth weight. These effects may be mediated by altered placental function. We hypothesized that placental function would be influenced by the gestational week of experiencing depressive symptoms and aimed to examine associations between maternal depressive symptoms during pregnancy and placental expression of genes involved in glucocorticoid and serotonin transfer between mother and fetus.nnnMETHODnWe studied women participating in a prospective pregnancy cohort: the Prediction and Prevention of Preeclampsia (PREDO) Study, Helsinki, Finland. Maternal depressive symptoms were assessed at 2-week intervals throughout pregnancy in 56 healthy women with singleton, term pregnancies. Messenger ribonucleic acid (mRNA) levels of glucocorticoid (GR) and mineralocorticoid (MR) receptors and serotonin transporter (SLC6A4), 11β-hydroxysteroid dehydrogenase type 1 (HSD1) and 2 (HSD2) were quantified in placental biopsies.nnnRESULTSnIn adjusted analyses women who reported higher depressive symptoms across the whole pregnancy had higher mRNA levels of GR [effect size 0.31 s.d. units, 95% confidence interval (CI) 0.01-0.60, pxa0=xa00.042] and MR (effect size 0.34 s.d. units, 95% CI 0.01-0.68, pxa0=xa00.047). These effects were significant for symptoms experienced in the third trimester of pregnancy for GR; findings for MR were also significant for symptoms experienced in the second trimester. GR and MR mRNA levels increased linearly by having the trimester-specific depressive symptoms scores 0, 1 or 2-3 times above the clinical cut-off for depression (pxa0=xa00.003, pxa0=xa00.049, respectively, and pxa0=xa00.004, pxa0=xa00.15 in adjusted analyses).nnnCONCLUSIONSnOur findings offer potential gestational-age-specific mechanisms linking maternal depressive symptoms during pregnancy via placental biology. Future studies will test whether these also link with adverse offspring outcomes.

Kisspeptin-10 inhibits angiogenesis in human placental vessels ex vivo and endothelial cells in vitro.

Recent studies suggest that kisspeptin (a neuropeptide central to the regulation of gonadotrophin secretion) has diverse roles in human physiology, including a putative role in implantation and placental function. Kisspeptin and its receptor are present in human blood vessels, where they mediate vasoconstriction, and kisspeptin is known to inhibit tumor metastasis and trophoblast invasion, both processes involving angiogenesis. We hypothesized that kisspeptin contributes to the regulation of angiogenesis in the reproductive system. The presence of the kisspeptin receptor was confirmed in human placental blood vessels and human umbilical vein endothelial cells (HUVEC) using immunochemistry. The ability of kisspeptin-10 (KP-10) (a shorter biologically active processed peptide) to inhibit angiogenesis was tested in explanted human placental arteries and HUVEC using complementary ex vivo and in vitro assays. KP-10 inhibited new vessel sprouting from placental arteries embedded in Matrigel and tube-like structure formation by HUVEC, in a concentration-dependent manner. KP-10 had no effect on HUVEC viability or apoptosis but induced concentration-dependent inhibition of proliferation and migration. In conclusion, KP-10 has antiangiogenic effects and, given its high expression in the placenta, may contribute to the regulation of angiogenesis in this tissue.

Maternal depressive symptoms during pregnancy, placental expression of genes regulating glucocorticoid and serotonin function and infant regulatory behaviors.

BACKGROUNDnGlucocorticoids and serotonin may mediate the link between maternal environment, fetal brain development and programming of offspring behaviors. The placenta regulates fetal exposure to maternal hormonal signals in animal studies, but few data address this in humans. We measured prospectively maternal depressive symptoms during pregnancy and mRNAs encoding key gene products determining glucocorticoid and serotonin function in term human placenta and explored associations with infant regulatory behaviors.nnnMETHODnBi-weekly self-ratings of the Center for Epidemiologic Studies Depression Scale from 12th to 13th gestational week onwards and term placental mRNAs of 11beta-hydroxysteroid dehydrogenase type 2 (HSD2B11), type 1 (HSD1B11), glucocorticoid (NR3C1), mineralocorticoid receptors (NR3C2) and serotonin transporter (SLC6A4) were obtained from 54 healthy mothers aged 32.2 ± 5.3 years with singleton pregnancies and without pregnancy complications. Infant regulatory behaviors (crying, feeding, spitting, elimination, sleeping and predictability) were mother-rated at 15.6 ± 4.2 days.nnnRESULTSnHigher placental mRNA levels of HSD2B11 [0.41 standard deviation (s.d.) unit increase per s.d. unit increase; 95% confidence interval (CI) 0.13-0.69, p = 0.005], HSD1B11 (0.30, 0.03-0.57, p = 0.03), NR3C1 (0.44, 0.19-0.68, p = 0.001) and SLC6A4 (0.26, 0.00-0.53, p = 0.05) were associated with more regulatory behavioral challenges of the infant. Higher placental NR3C1 mRNA partly mediated the association between maternal depressive symptoms during pregnancy and infant regulatory behaviors (p < 0.05).nnnCONCLUSIONSnHigher placental expression of genes regulating feto-placental glucocorticoid and serotonin exposure is characteristic of infants with more regulatory behavioral challenges. Maternal depression acts, at least partly, via altering glucocorticoid action in the placenta to impact on offspring regulatory behaviors.

Associations between maternal level of education and occupational status with placental glucocorticoid regeneration and sensitivity

Low socio‐economic status (SES) is associated with increased disease risk in the involved and the next generation. The effects of low maternal SES on the offspring may be initiated prenatally. We hypothesized that fetoplacental glucocorticoid exposure might mediate the links. We examined associations between maternal level of education and occupational status (used as indices of SES) and placental expression of genes involved in glucocorticoid exposure and transfer between the mother and foetus.

Association Between Excessive Daytime Sleepiness and Severe Hypoglycemia in People With Type 2 Diabetes The Edinburgh Type 2 Diabetes Study

OBJECTIVE Sleep-disordered breathing and sleepiness cause metabolic, cognitive, and behavioral disturbance. Sleep-disordered breathing is common in type 2 diabetes, a condition that requires adherence to complex dietary, behavioral, and drug treatment regimens. Hypoglycemia is an important side effect of treatment, causing physical and psychological harm and limiting ability to achieve optimal glycemic control. We hypothesized that sleep disorder might increase the risk of hypoglycemia through effects on self-management and glucose regulation. RESEARCH DESIGN AND METHODS People with type 2 diabetes (n = 898) completed questionnaires to assess sleep-disordered breathing, daytime sleepiness, and occurrence of severe hypoglycemia. RESULTS Subjects who scored highly on the Epworth Sleepiness Scale were significantly more likely to have suffered from severe hypoglycemia. This was a significant predictor of severe hypoglycemia in regression analysis including the variables age, sex, duration of diabetes, HbA1c, BMI, and treatment type. CONCLUSIONS Daytime sleepiness may be a novel risk factor for hypoglycemia.

Physical activity and hypothalamic–pituitary–adrenocortical axis function in adolescents

Little is known about the associations between physical activity (PA) and hypothalamic-pituitary-adrenocortical axis (HPAA) activity in adolescents. This knowledge could offer insight into the links between PA and well-being in youth. We studied whether objectively-measured PA is associated with diurnal salivary cortisol responses and morning salivary cortisol responses after a low-dose overnight dexamethasone suppression test (DST) in adolescent girls and boys. We conducted a cross-sectional birth cohort study in Helsinki, Finland. At a mean age of 12.4 (SD=0.5) years, 150 girls and 133 boys wore wrist-worn accelerometers over at least 4 days to measure PA. Their salivary cortisol was measured across 1 day and upon awakening after a low-dose overnight DST (3 μg/kg of weight). Girls with higher overall PA and vigorous PA (VPA), and less sedentary time had lower salivary cortisol upon awakening and/or after (decreases between |0.17| and |0.25| SDs per SD increase in overall PA, VPA and decrease in sedentary time; P-values<0.039). Boys with higher overall PA, and less sedentary time had greater suppression of salivary cortisol following overnight DST (suppression between |0.24| and |0.27| SDs per SD increase in overall PA and decrease in sedentary time; P-values<0.012). Overall PA, VPA and sedentary time did not associate with DST suppression in girls or with diurnal salivary cortisol in boys. These results show that PA is associated with altered HPAA function in early adolescents, and that the associations are sex specific.

Altered maternal hypothalamic-pituitary-adrenal axis activity in obese pregnancy is associated with macrosomia and prolonged pregnancy

BACKGROUNDnThe hypothalamic-pituitary-adrenal (HPA) axis is important for fetal growth and timing of parturition. Maternal obesity is associated with macrosomia (birthweight ⩾4000g) and prolonged pregnancy (⩾41weeks). We aimed to characterise HPA axis hormones in obese pregnancy and to test associations with these pregnancy outcomes.nnnMETHODnFasting cortisol was measured by radioimmunoassay in venous blood at 16, 28 and 36 weeks of gestation in 286 obese (BMI 44.05±3.98kg/m(2)) and 137 lean (BMI 22.71±1.66kg/m(2)) pregnant women. In subsets (n=20 obese, 20 lean) we measured corticosteroid binding globulin (CBG) and CRH by radioimmunoassay; progesterone, estradiol (E2), estriol (E3) and sex-hormone-binding-globulin (SHBG) by ELISA; and albumin by bromocresol green binding. Free cortisol levels were calculated using Coolens equation.nnnRESULTSnCortisol, CBG, calculated free cortisol, CRH, E2, E3, progesterone and SHBG levels rose similarly during pregnancy in obese and lean, but were significantly lower in obese (p<0.05). In obese, lower free cortisol at 16 weeks was associated with higher birthweight (r=-0.46, p<0.05). Cortisol was not associated with labour onset. CRH was significantly lower at 36 weeks in women who delivered at ⩾41weeks and in women with macrosomic babies (p<0.05); and correlated negatively with gestation at delivery in obese (r=-0.557, p<0.05).nnnCONCLUSIONnOur findings suggest that decreased HPA axis activity in obese pregnancy may be a mechanism underlying macrosomia and prolonged pregnancy.

Additive effects of maternal depressive symptoms during pregnancy and three years after childbirth on offspring psychiatric symptoms in early childhood

Introduction: Themechanisms underlying the effects ofmaternal prenatal stress on foetal development remain elusive. One hypothesis is that maternal stress leads to increased cortisol blood concentrations, which in turn may overstimulate the secretion of placental corticotropin releasing hormone (CRH) into the foetal circulation. However, whether acute stress-induced rises in maternal cortisol affect foeto-placental CRH levels is unknown. We therefore aimed at exploring the association between the acutematernal psychobiological stress response as well as chronic maternal stress perception and foeto-placental CRH levels. Methods: We examined thirty-four healthy pregnant women (37.4±4.0 years) undergoing second-trimester amniocentesis. The amniocentesis served as a naturalistic stress situationduringwhich we repeatedly obtained questionnaires on maternal acute stress perception and saliva samples for cortisol analysis. We furthermore monitored maternal cardiac activity continuously (i.e., heart rate, heart rate variability). Thewomen also responded to the social overload scale of the Trier Inventory of Chronic Stress (Schultz and Schlotz, 1999). CRH levels were analysed from an amniotic fluid aliquot. Results: The amniocentesis induced significant changes in maternal stress perception, salivary cortisol, and cardiac activity. No significant association between the acute psychobiological stress response and amniotic CRH was apparent (all p> .10). However, maternal chronic social overload was positively correlated with CRH levels (Kendall’s tau= .34, p= .02). Conclusions: The current findings provide evidence thatmaternal chronic stress affects placental CRH secretion. However, we cannot exclude that a different or longer timeframe might be required to detect an association between acute maternal stress and foeto-placental CRH. Further studies are called for.