Hannah K. Palliser

Neurosteroids in the fetus and neonate: Potential protective role in compromised pregnancies

Complications during pregnancy and birth asphyxia lead to brain injury, with devastating consequences for the neonate. In this paper we present evidence that the steroid environment during pregnancy and at birth aids in protecting the fetus and neonate from asphyxia-induced injury. Earlier studies show that the placental progesterone production has a role in the synthesis and release of neuroactive steroids or their precursors into the fetal circulation. Placental precursor support leads to remarkably high concentrations of allopregnanolone in the fetal brain and to a dramatic decline with the loss of the placenta at birth. These elevated concentrations influence the distinct behavioral states displayed by the late gestation fetus and exert a suppressive effect that maintains sleep-like behavioral states that are present for much of fetal life. This suppression reduces CNS excitability and suppresses excitotoxicity. With the availability of adequate precursors, mechanisms within the fetal brain ultimately control neurosteroid levels. These mechanisms respond to episodes of acute hypoxia by increasing expression of 5alpha-reductase and P450scc enzymes and allopregnanolone synthesis in the brain. This allopregnanolone response, and potentially that of other neurosteroids including 5alpha-tetrahydrodeoxycorticosterone (TH-DOC), reduces hippocampal cell death following acute asphyxia and suggests that stimulation of neurosteroid production may protect the fetal brain. Importantly, inhibition of neurosteroid synthesis in the fetal brain increases the basal cell death suggesting a role in controlling developmental processes late in gestation. Synthesis of neurosteroid precursors in the fetal adrenal such as deoxycorticosterone (DOC), and their conversion to active neurosteroids in the fetal brain may also have a role in neuroprotection. This suggests that the adrenal glands provide precursor DOC for neurosteroid synthesis after birth and this may lead to a switch from allopregnanolone alone to neuroprotection mediated by allopregnanolone and TH-DOC.

Sex-dependent effect of a low neurosteroid environment and intrauterine growth restriction on foetal guinea pig brain development

Progesterone and its neuroactive metabolite, allopregnanolone, are present in high concentrations during pregnancy, but drop significantly following birth. Allopregnanolone influences foetal arousal and enhances cognitive and behavioural recovery following traumatic brain injury. Inhibition of allopregnanolone synthesis increases cell death in foetal animal brains with experimental hypoxia. We hypothesised that complications during pregnancy, such as early or preterm loss of placental steroids and intrauterine growth restriction (IUGR), would disrupt the foetal neurosteroid system, contributing to poor neurodevelopmental outcomes. This study aimed to investigate the effects of chronic inhibition of allopregnanolone synthesis before term and IUGR on developmental processes in the foetal brain. Guinea pig foetuses were experimentally growth restricted at mid-gestation and treated with finasteride, an inhibitor of allopregnanolone synthesis. Finasteride treatment reduced foetal brain allopregnanolone concentrations by up to 75% and was associated with a reduction in myelin basic protein (MBP) (P = 0.001) and an increase in glial fibrillary acidic protein expression in the subcortical white matter brain region (P < 0.001). IUGR resulted in decreased MBP expression (P < 0.01) and was associated with a reduction in the expression of steroidogenic enzyme 5α-reductase (5αR) type 2 in the foetal brain (P = 0.061). Brain levels of 5αR1 were higher in male foetuses (P = 0.008). Both IUGR and reduced foetal brain concentrations of allopregnanolone were associated with altered expression of myelination and glial cell markers within the developing foetal brain. The potential role of neurosteroids in protecting and regulating neurodevelopmental processes in the foetal brain may provide new directions for treatment of neurodevelopmental disorders in infants who are exposed to perinatal insults and pathologies.

The effect of betamethasone treatment on neuroactive steroid synthesis in a foetal Guinea pig model of growth restriction.

There are ongoing concerns that antenatal corticosteroids, which are administered to women at high risk of delivering preterm to reduce the incidence of respiratory distress syndrome, have adverse effects on foetal brain development and subsequent effects on behaviour and learning, when administered as repeated courses. The present study aimed to examine whether repeated betamethasone treatment alters the expression of the key‐rate limiting enzyme, 5α‐reductase, in the synthetic pathway of the potent neuroactive steroid allopregnanolone in the brain and placenta and whether this effect is potentiated in growth restricted foetuses. To investigate this, pregnant guinea pigs carrying either control (sham surgery) or growth‐restricted foetuses were treated with vehicle or betamethasone (1 mg/kg/day) for 4 days prior to sacrifice (65d). Placental insufficiency was induced by the ablation of uterine artery branches supplying each placenta at mid gestation, resulting in foetal growth restriction characterised by ‘brain sparing’. Real‐time reverse transcriptase polymerase chain reaction was used to determine relative 5α‐reductase type 1 and 2 mRNA expression in the placenta and brain. Immunohistochemistry was used to examine the glial fibrillary acidic protein (GFAP) expression in the subcortical white matter, CA1 and dentate regions of the hippocampus. 5α‐reductase type 2 mRNA expression in the brain was markedly reduced by betamethasone treatment in male foetuses compared to vehicle‐treated controls but not in female foetuses. In addition, 5α‐reductase type 1 expression in the brain was increased by growth restriction and/or betamethasone treatment in female foetuses but expression in males foetuses did not increase. 5α‐reductase type 2 expression in the placenta was markedly reduced by betamethasone treatment compared to vehicle‐treated control. Intrauterine growth restriction and betamethasone treatment reduced GFAP expression in the CA1 region of the hippocampus in the brains of male but not female foetuses. These data indicate that betamethasone treatment suppresses placental expression and has sexually dimorphic effects on expression of neuroactive steroid synthetic enzymes in the brain. These actions may lead to adverse effects on the developing brain, particularly in male foetuses, such as the observed effects on GFAP expression.

Changes in human placental 5α-reductase isoenzyme expression with advancing gestation: effects of fetal sex and glucocorticoid exposure

5alpha-reduced steroids, including allopregnanolone, suppress neuronal activity and can have neuroprotective effects in the fetus. 5alpha-reductases in the placenta may contribute precursors to brain allopregnanolone synthesis. Preterm birth and glucocorticoids, administered for fetal lung maturation or for maternal asthma, may influence reductase expression. The aims of the present study were to evaluate placental 5alpha-reductase isoform expression during late gestation and to examine fetal sex differences and the effects of glucocorticoid therapies on the expression of these enzymes. Expression of the two 5alpha-reductase isoenzymes was measured in placental samples, whereas cortisol concentrations were measured in cord blood, from two cohorts. The first cohort consisted of women who delivered preterm and received betamethasone treatment (n=41); the second cohort consisted of women who delivered at term and were either healthy controls (n=30) or asthmatics who had used glucocorticoids (n=24). Placental expression of both isoenzymes increased with advancing gestation and there were marked sex differences in levels of 5alpha-reductase I (P<0.05), but not of 5alpha-reductase II. The expression of both enzymes was positively correlated with cortisol levels (P<0.05), but there was no effect of recent glucocorticoid exposure. These findings suggest that the preterm neonate may have lower developmental exposure to 5alpha-reduced steroids and may lack steroid-mediated neuroprotection depending on fetal sex.

Stress in pregnancy: a role for neuroactive steroids in protecting the fetal and neonatal brain

Stressors during pregnancy can lead to perinatal brain injury resulting in serious neurological impairment. Neuroactive steroid concentrations are elevated during pregnancy and are remarkably high in the fetal brain. In long-gestation species, including humans, these steroids enhance GABAergic inhibition and reduce the possibility of cerebral excitotoxicity during the last third of gestation. The fetal brain responds to acute hypoxia/ischemia by increasing steroid concentrations further as protection against excitotoxic cell death. The placenta has a key role in maintaining neuroactive steroid concentrations in the brain by acting as a source of precursors for neuroactive steroid synthesis. Gestational neuroactive steroid concentrations are needed for normal cell proliferation and cell death in the late gestation brain and a loss of these steroids at preterm birth may adversely affect development and vulnerability to injury.

Changes in Neuroactive Steroid Concentrations After Preterm Delivery in the Guinea Pig

Background: Preterm birth is a major cause of neurodevelopmental disorders. Allopregnanolone, a key metabolite of progesterone, has neuroprotective and developmental effects in the brain. The objectives of this study were to measure the neuroactive steroid concentrations following preterm delivery in a neonatal guinea pig model and assess the potential for postnatal progesterone replacement therapy to affect neuroactive steroid brain and plasma concentrations in preterm neonates. Methods: Preterm (62-63 days) and term (69 days) guinea pig pups were delivered by cesarean section and tissue was collected at 24 hours. Plasma progesterone, cortisol, allopregnanolone, and brain allopregnanolone concentrations were measured by immunoassay. Brain 5α-reductase (5αR) expression was determined by Western blot. Neurodevelopmental maturity of preterm neonates was assessed by immunohistochemistry staining for myelination, glial cells, and neurons. Results: Brain allopregnanolone concentrations were significantly reduced after birth in both preterm and term neonates. Postnatal progesterone treatment in preterm neonates increased brain and plasma allopregnanolone concentrations. Preterm neonates had reduced myelination, low birth weight, and high mortality compared to term neonates. Brain 5αR expression was also significantly reduced in neonates compared to fetal expression. Conclusions: Delivery results in a loss of neuroactive steroid concentrations resulting in a premature reduction in brain allopregnanolone in preterm neonates. Postnatal progesterone therapy reestablished neuroactive steroid levels in preterm brains, a finding that has implications for postnatal growth following preterm birth that occurs at a time of neurodevelopmental immaturity.

Prenatal Stress Alters Hippocampal Neuroglia and Increases Anxiety in Childhood

Prenatal stress has been associated with detrimental outcomes of pregnancy, including altered brain development leading to behavioural pathologies. The neurosteroid allopregnanolone has been implicated in mediating some of these adverse outcomes following prenatal stress due to its potent inhibitory and anxiolytic effects on the brain. The aims of the current study were to characterise key markers for brain development as well as behavioural parameters, adrenocortical responses to handling and possible neurosteroid influences towards outcomes in guinea pig offspring in childhood. Pregnant guinea pig dams were exposed to strobe light for 2 h (9-11 a.m.) on gestational days 50, 55, 60, and 65 and were left to deliver spontaneously at term and care for their litter. Behavioural testing (open-field test, object exploration test) of the offspring was performed at postnatal day 18 (with salivary cortisol and DHEA measured), and brains were collected at post-mortem on day 21. Markers of brain development myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) were assessed via immunohistochemistry, and the neurosteroid allopregnanolone and its rate-limiting enzymes 5α-reductase types 1 and 2 (5αR1/2) were measured in neonatal brains by radioimmunoassay, reverse transcriptase polymerase chain reaction (RT-PCR), and Western blot, respectively. Brain-derived neurotrophic factor protein was measured as a marker of synaptic plasticity, and GABAA receptor subunit expression was also assessed using RT-PCR. Neonates born from mothers stressed during late pregnancy showed a reduction in both MBP (p < 0.01) and GFAP (p < 0.05) expression in the CA1 region of the hippocampus at 21 days of age. Pups of prenatally stressed pregnancies also showed higher levels of anxiety and neophobic behaviours at the equivalent of childhood (p < 0.05). There were no significant changes observed in allopregnanolone levels, 5αR1/2 expression, or GABAA receptor subunit expression in prenatally stressed neonates compared to controls. This study shows alterations in markers of myelination and reactive astrocytes in the hippocampus of offspring exposed to prenatal stress. These changes are also observed in offspring that show increased anxiety behaviours at the equivalent of childhood, which indicates ongoing structural and functional postnatal changes after prenatal stress exposure.

Effects of Prenatal Stress on Fetal Neurodevelopment and Responses to Maternal Neurosteroid Treatment in Guinea Pigs

Background: Maternal psychosocial stress during pregnancy is associated with adverse neonatal outcomes. These outcomes result from changes in fetal brain development and lead to disrupted cognitive, behavioural and emotional development. The neurosteroid allopregnanolone has been shown to reduce neural excitability and aid in protecting the fetal brain from excitotoxic insults. The objectives of this study were to assess the effect of prenatal maternal stress on fetal brain development with and without maternal allopregnanolone treatment. Methods: Pregnant guinea pigs were subjected to stress induced by exposure to a strobe light at 50, 55, 60 and 65 days gestation. Salivary cortisol levels were measured before and after each exposure. Fetal brains were assessed for markers of brain development using immunohistochemistry and plasma allopregnanolone was measured by radioimmunoassay. Results: Female, but not male prenatal stress-exposed fetuses demonstrated higher brain-to-liver ratios (BLR). Male fetuses showed significantly reduced expression of myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and both males and females showed reduced expression of microtubule-associated protein 2 (MAP2). These markers were not affected by maternal allopregnanolone treatment. However, maternal allopregnanolone treatment resulted in an increase in fetal plasma allopregnanolone concentrations in control pregnancies but concentrations were not raised after prenatal stress exposure. Conclusions: These findings indicate that the effects of prenatal stress on fetal brain development are sexually dimorphic with more pronounced negative effects seen on male neurodevelopment. Allopregnanolone treatment was not effective in raising fetal plasma concentrations after prenatal stress suggesting a stress-induced dysregulation of neurosteroid pathways during gestation. Interestingly, this study directly implicates prenatal stress in the disruption of fetal neurosteroid levels, such that it may mediate some of the deleterious effects on fetal neurodevelopment by facilitating a deficit in normal endogenous neuroprotective mechanisms.

Prostaglandin E and F receptor expression and myometrial sensitivity at labor onset in the sheep

Abstract Prostaglandins (PGs) play a pivotal role in the initiation and progression of term and preterm labor. Uterine activity is stimulated primarily by PGE2 and PGF2α acting on prostaglandin E (EP) and prostaglandin F (FP) receptors, respectively. Activation of FP receptors strongly stimulates the myometrium, whereas stimulation of EP receptors may lead to contraction or relaxation, depending on the EP subtype (EP1–4) expression. Thus, the relative expression of FP and EP1–4 may determine the responsiveness to PGE2 and PGF2α. The aims of this study were to characterize the expression of EP1–4 and FP in intrauterine tissues and placentome, together with myometrial responsiveness to PG, following the onset of dexamethasone-induced preterm and spontaneous term labor. Receptor mRNA expression was measured using quantitative real-time polymerase chain reaction using species-specific primers. There was no increase in myometrial contractile receptor expression at labor onset, nor was there a change in sensitivity to PGE2 and PGF2α. This suggests expression of these receptors reaches maximal levels by late gestation in sheep. Placental tissue showed a marked increase in EP2 and EP3 receptor expression, the functions of which are unknown at this time. Consistent with previous reports, these results suggest that PG synthesis is the main factor in the regulation of uterine contractility at labor. This is the first study to simultaneously report PG E and F receptor expression in the key gestational tissues of the sheep using species-specific primers at induced-preterm and spontaneous labor onset.

Pathological interactions with the timing of birth and uterine activation

The physiological processes that regulate the onset of parturition and birth are slowly being elucidated, and the points at which pathology can intervene are becoming more apparent. The data support the view that multiple pathways lead to myometrial activation. The clinical corollary is that combinations of tocolytics that operate via different mechanisms may be more effective than single agents. It may also be necessary to divide preterm labour into groups based on underlying mechanisms and to tailor therapy accordingly.