Effects of combined IUGR and prenatal stress on the development of the hippocampus in a fetal guinea pig model.

Intrauterine growth restriction (IUGR) and maternal stress during pregnancy are two compromises that negatively impact neurodevelopment and increase the risk of developing later life neuropsychiatric disorders such as schizophrenia, depression and behavioural disorders. Neurosteroids, particularly allopregnanolone, are important in protecting the developing brain and promoting many essential neurodevelopmental processes. Individually, IUGR and prenatal stress (PS) reduce myelination and neurogenesis within affected fetal brains, however less information is available on the combined effects of these two disorders on the term fetal brain. This study aimed to investigate how IUGR and PS impairs the neurosteroid pathway when combined using a guinea pig model, and how these then disrupt the neurodevelopment of the fetus. Uterine artery blood flow restriction was performed at GA30-35 to induce growth restriction, whilst PS was induced by exposure of the dam to a strobe light during gestation commencing GA40 and repeated every 5 days. Exposure in this model caused reductions in hippocampal CA1 MBP immunostaining of male fetuses in both IUGR alone and IUGR+PS paradigms but only by IUGR in the subcortical white mater, compared with control males. Plasma allopregnanolone was reduced by both stressors irrespective of sex, whereas GFAP or MAP2 expression were not affected by either stressor. Female neurodevelopment, as assessed by these markers, was unimpeded by these compromises. The addition of prenatal stress did not further compound these deficits.

Effect of postnatal progesterone therapy following preterm birth on neurosteroid concentrations and cerebellar myelination in guinea pigs.

Allopregnanolone protects the fetal brain and promotes normal development including myelination. Preterm birth results in the early separation of the infant from the placenta and consequently a decline in blood and brain allopregnanolone concentrations. Progesterone therapy may increase allopregnanolone and lead to improved oligodendrocyte maturation. The objectives of this study were to examine the efficacy of progesterone replacement in augmenting allopregnanolone concentrations during the postnatal period and to assess the effect on cerebellar myelination - a region with significant postnatal development. Preterm guinea pig neonates delivered at 62 days of gestation by caesarean section received daily s.c. injections of vehicle (2-Hydroxypropyl-ß-cyclodextrin) or progesterone (16 mg/kg) for 8 days until term-equivalent age (TEA). Term delivered controls (PND1) received vehicle. Neonatal condition/wellbeing was scored, and salivary progesterone was sampled over the postnatal period. Brain and plasma allopregnanolone concentrations were measured by radioimmunoassay; cortisol and progesterone concentrations were determined by enzyme immunoassay; and myelin basic protein (MBP), proteolipid protein (PLP), oligodendroctye transcription factor 2 (OLIG2) and platelet-derived growth factor receptor-α (PDGFRα) were quantified by immunohistochemistry and western blot. Brain allopregnanolone concentrations were increased in progesterone-treated neonates. Plasma progesterone and cortisol concentrations were elevated in progesterone-treated male neonates. Progesterone treatment decreased MBP and PLP in lobule X of the cerebellum and total cerebellar OLIG2 and PDGFRα in males but not females at TEA compared with term animals. We conclude that progesterone treatment increases brain allopregnanolone concentrations, but also increases cortisol levels in males, which may disrupt developmental processes. Consideration should be given to the use of non-metabolizable neurosteroid agonists.

Preterm birth affects GABAA receptor subunit mRNA levels during the foetal-to-neonatal transition in guinea pigs.

Modulation of gamma-aminobutyric acid A (GABAA) receptor signalling by the neurosteroid allopregnanolone has a major role in late gestation neurodevelopment. The objective of this study was to characterize the mRNA levels of GABAA receptor subunits (α4, α5, α6 and δ) that are key to neurosteroid binding in the brain, following preterm birth. Myelination, measured by the myelin basic protein immunostaining, was used to assess maturity of the preterm brains. Foetal guinea pig brains were obtained at 62 days' gestational age (GA, preterm) or at term (69 days). Neonates were delivered by caesarean section, at 62 days GA and term, and maintained until tissue collection at 24 h of age. Subunit mRNA levels were quantified by RT-PCR in the hippocampus and cerebellum of foetal and neonatal brains. Levels of the α6 and δ subunits were markedly lower in the cerebellum of preterm guinea pigs compared with term animals. Importantly, there was an increase in mRNA levels of these subunits during the foetal-to-neonatal transition at term, which was not seen following preterm birth. Myelination was lower in preterm neonatal brains, consistent with marked immaturity. Salivary cortisol concentrations, measured by EIA, were also higher for the preterm neonates, suggesting greater stress. We conclude that there is an adaptive increase in the levels of mRNA of the key GABAA receptor subunits involved in neurosteroid action after term birth, which may compensate for declining allopregnanolone levels. The lower levels of these subunits in preterm neonates may heighten the adverse effect of the premature decline in neurosteroid exposure.

Increased placental neurosteroidogenic gene expression precedes poor outcome in the preterm guinea pig.

Placental 5α-reductase (5αR) is influenced by in utero compromises and has a role in regulating neuroactive steroid concentrations in the fetus. The objective of this study was to determine if changes in placental 5αR were associated with neonatal outcome after birth. Guinea pigs were delivered by cesarean section at term (GA69, n=22) or preterm (GA62, n=36) and the placenta collected. Preterm neonates were maintained for 24 h unless their condition deteriorated before this time. Enzyme mRNA expression of 5αR type-1 and 5αR type-2 were determined using real-time PCR. All preterm neonates had significantly higher 5αR2 expression in their placenta compared with placentae from term neonates (P<0.0001). Expression was also markedly higher in the placentae from neonates that did not survive until 24 h, compared with surviving preterm neonates (P=0.04). These findings suggest differences of in utero neurosteroidogenic capacity between surviving and non-surviving preterm guinea pig neonates. The increased 5αR2 mRNA expression in the placenta of non-survivors suggests an induction of the neurosteroid pathway due to prior exposure to an in utero compromise, with such exposure possibly a predisposing factor that contributed to their poor ex utero outcome.

Progesterone receptor isoform expression in response to in utero growth restriction in the fetal guinea pig brain.

Intra-uterine growth restriction (IUGR) is a significant in utero complication that can have profound effects on brain development including reduced myelination and deficits that can continue into adulthood. Progesterone increases oligodendrocyte proliferation and myelin expression, an action that may depend on the expression of progesterone receptor (PR) isoforms A (PRA) and B (PRB). The objective of this study was to determine the effect of IUGR on PR isoform expression in the brain of male and female fetuses and whether effects were associated with a reduction in myelination. We used a guinea pig model that involves selective reduction in maternal perfusion to the placenta at midgestation (35 days, term 70 days). This resulted in a significant reduction in body weight with marked sparing of brain weight. PRA, PRB and myelin basic protein (MBP) expression were measured in the brains of male and female growth-restricted and control fetuses at late gestation. MBP, as a measure of myelination, was found to decrease in association with IUGR in the CA1 hippocampal region with no change observed in the cortical white matter. There was a marked increase in PRA, PRB and total PR expression in the IUGR fetal brain. Control female fetuses demonstrated significantly higher PRA:PRB ratios than males; however, this sex difference was abolished with IUGR. These data suggest the central nervous system effects of clinical use of progesterone augmentation therapy in late pregnancy should be carefully evaluated. The overall upregulation of PR isoforms in association with IUGR suggests increased progesterone action and a possible neuroprotective mechanism.

Molecular characterization of renin-angiotensin system components in human intrauterine tissues and fetal membranes from vaginal delivery and cesarean section.

A prorenin-angiotensin system (RAS) could, via the (pro)renin receptor (ATP6AP2), have various effects in human intrauterine tissues, either directly by prorenin/ATP6AP2 cell signaling, or indirectly via angiotensin II and/or angiotensin 1-7. Here we describe RAS components in fetal membranes, decidua and placenta collected at elective cesarean section (non-laboring), after spontaneous delivery (after labor, n = 38), and in myometria (n = 16) from elective (non-laboring) or emergency cesarean (laboring) deliveries. Angiotensinogen (AGT), angiotensin-converting enzyme 1 and 2 (ACE; ACE2), angiotensin receptor 1 and 2 (AGTR1; AGTR2) and angiotensin 1-7 receptor (MAS1) mRNAs were measured by qRT-PCR and proteins were localized by immunohistochemistry. In myometrium, prorenin (REN), ATP6AP2, and downstream signaling proteins zinc finger and BTB domain-containing protein 16 (ZBTB16), transforming growth factor-ß1 (TGFß1) and prostaglandin-endoperoxide synthase 2 (PTGS2) mRNAs were also measured. RAS mRNAs, except AGTR1 and AGTR2, were abundant in decidua and lowest in amnion compared to the other tissues. ACE, AGT and PTGS2 mRNAs were higher in laboring than non-laboring myometrium, suggesting that the myometrial RAS is involved in labor. Angiotensinogen and prorenin staining in amnion, chorion and decidua was pervasive despite their mRNAs being low in amnion and chorion. In placenta, prorenin, angiotensinogen and AGTR2 were present in syncytiotrophoblasts, ACE was in fetal endothelium, while ACE2 distribution was diffuse. AGTR1 and AGTR2 mRNAs and proteins were abundant. No differences were evident in the staining patterns with labor. These results are consistent with the hypothesis that fetal vascular ACE might contribute angiotensin II to the fetus, whilst syncytial ACE2 might hypothetically have a role in converting angiotensin II to angiotensin 1-7 in maternal blood.

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, 5alpha-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 5alpha-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. 5alpha-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, 5alpha-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. 5alpha-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.

Role of neurosteroids in regulating cell death and proliferation in the late gestation fetal brain.

The neurosteroid allopregnanolone (AP) is a GABAergic agonist that suppresses central nervous system (CNS) activity in the adult brain, and by reducing excitotoxicity is considered to be neuroprotective. A role for neurosteroids in the developing brain, particularly in late gestation, is still debated. The aim of this study was to investigate effects on proliferation and cell death in the brain of late gestation fetal sheep after inhibition of AP synthesis using finasteride, a 5alpha-reductase type 2 (5alpha-R2) inhibitor. Catheters were implanted in fetal sheep at approximately 125 days of gestation. At 3-4 days postsurgery, fetuses received infusions of either finasteride (20 mg/kg/h; n=5), the AP analogue alfaxalone (5 mg/kg/h; n=5), or finasteride and alfaxalone together (n=5). Brains were obtained at 24 h after infusion to determine cell death (apoptotic or necrotic) and cell proliferation in the hippocampus and cerebellum, areas known to be susceptible to excitotoxic damage. Finasteride treatment significantly increased apoptosis (activated caspase-3 expression) in hippocampal CA3 and CA1, and cerebellar molecular and granular layers, an effect abolished by co-infusion of alfaxalone and finasteride. Double-label immunohistochemistry showed that both neurons and astrocytes were caspase-3 positive. Finasteride treatment also increased the number of dead (pyknotic) cells in the hippocampus and cerebellum (Purkinje cells), but not when finasteride+alfaxalone was infused. Cell proliferation (Ki-67-immunoreactivity) increased after finasteride treatment; double-labeling showed the majority of Ki-67-positive cells were astrocytes. Thus, steroids such as AP appear to influence the constitutive rate of apoptosis and proliferation in the hippocampus and cerebellum of the fetal brain, and suggest an important role for neurosteroids in the development of the brain.

Inhibition of neurosteroid synthesis increases asphyxia-induced brain injury in the late gestation fetal sheep.

Allopregnanolone (AP) is a potent GABAergic agonist that suppresses CNS activity, seizure threshold, and excitotoxicity in the adult brain. AP is present in the fetal sheep brain and increases rapidly after asphyxial insult due to increased 5alpha-reductase type-2 (5alphaR-2) expression. The aim of this study was to use finasteride to suppress fetal neurosteroid synthesis, and then determine the effect on brain injury, particularly in the hippocampus, of asphyxia induced in utero by brief occlusion of the umbilical cord. Catheters and an inflatable umbilical cord cuff were implanted in fetal sheep at approximately 125 days gestation. Five days later the fetuses received either finasteride (20 mg/kg/h) or vehicle (40% hydroxypropyl-beta-cyclodextrin) for 2 h. The umbilical cord was occluded (UCO) for 5 min at 30 min after starting the infusion. The fetal brain was obtained 24 h later for examination of activated caspase-3 expression as an index of apoptosis, and to measure AP content. Finasteride treatment alone significantly reduced AP content and increased the number of caspase-3 positive cells in the hippocampus, cerebellum, and the subcallosal bundle, indicating that AP modulates the normal rate of apoptosis in the developing brain. UCO in vehicle and finasteride-treated fetuses produced a similar, marked decrease in O2 saturation (5.8+/-0.6%), but after finasteride treatment UCO caused a significantly greater increase in the number of caspase-3 positive cells in the hippocampal cornu ammonis 3 (CA3) (57.3+/-1.6%) compared with the vehicle-treated fetuses. Thus, 5alpha-reduced steroids such as AP may be protective in reducing cell death following acute fetal asphyxia. Perturbation of normal fetal neurosteroid levels in late gestation (e.g. due to preterm birth, or maternal synthetic steroid treatment to induce fetal lung maturation) could adversely affect brain development and increase its vulnerability to injury.

Changes in the expression of prostaglandin E and F synthases at induced and spontaneous labour onset in the sheep.

The differential production of prostaglandin (PG) F(2 alpha) and PGE(2) within the uterine compartment may play a role in controlling myometrial contraction. We hypothesized that the enzymes downstream of PG endoperoxide synthase-2 (PGHS-2) determine the ratio of PGF(2 alpha) and PGE(2) in the utero-ovarian vein plasma and the time of normal and preterm labour onset. The aim of this study was to simultaneously determine the expression of PGF and PGE synthases (PGFS and PGES) in gestational tissues at spontaneous and induced-preterm labour in sheep. Myometrial, endometrial and placental tissue were obtained from ewes in dexamethasone-induced preterm labour, age-matched control ewes, and ewes in spontaneous term labour for analysis of mRNA expression by real-time PCR. PGFS mRNA expression was significantly increased following dexamethasone-induced and spontaneous labour onset in placentome (P<0.01) but was unchanged in the myometrium and endometrium. In contrast, PGES mRNA expression remained unchanged or decreased. PGHS-2 mRNA expression was increased in all tissues examined in both dexamethasone-induced and spontaneous labour (P<0.001). Plasma PGE(2) and PGF(2 alpha) concentrations rose in both dexamethasone-induced and spontaneous labour with the ratio of PGF(2 alpha):PGE(2) increased with labour onset (P<0.05). These results are consistent with the hypothesis that the increased expression, of PGFS is responsible for the increased PGF(2 alpha):PGE(2) ratio and this, together with increased PGHS-2 expression, accounts for myometrial activity at labour onset. The findings point to PGFS expression as a key factor in regulating the uterotonic process in the sheep.

Progesterone receptor localization and isoforms in myometrium, decidua, and fetal membranes from rhesus macaques: evidence for functional progesterone withdrawal at parturition.

OBJECTIVE: It is not known whether withdrawal of progesterone (P) action is a prerequisite for parturition in women or in nonhuman primates because concentrations of circulating progesterone or progesterone receptors (PR) in myometrium and decidua do not decrease before delivery. To examine this potentially important regulatory mechanism, we determined PR isoforms, PR localization, and mRNA in myometrium, decidua, and fetal membranes from rhesus monkeys during pregnancy and in spontaneous labor at term. METHODS: Gestational tissues were obtained midpregnancy (day 80-100), late pregnancy (day 130-145), and during spontaneous labor at term (day 161-167). Samples of rhesus monkey myometrium, decidua, chorion-decidua, and amnion were collected and analyzed for total nuclear and cytosolic PR by competitive binding assay. Progesterone receptor isoforms were identified and quantified by Western blot analysis, and PR mRNA was determined by a specific ribonuclease protection assay. Nuclear PR was localized by immunohistochemistry with monoclonal anti-PR (JZB39) after microwave stabilization. RESULTS: Myometrium and decidua showed no change in total PR during pregnancy and labor. Nuclear PR was not detected in fetal membranes by binding assay but was localized in amnion epithelial and mesenchymal cells and in chorion laeve cytotrophoblasts by immunohistochemistry. Staining for PR was substantially less by serial antibody dilution in fetal membranes than in decidua. Message for PR was confirmed in all tissues analyzed. A significant (P <.05) shift in the ratio of PR isoforms (from PR-B dominance at midpregnancy to PR-A dominance in labor) was observed in myometrium but not in decidua. Both PR-A and PR-B isoforms and PR nuclear staining were nearly undetectable in amnion obtained during labor. CONCLUSION: A shift to PR-A dominance in myometrium at term together with a loss of PR in fetal membranes provides evidence for a functional progesterone withdrawal mechanism, which may facilitate the initiation of parturition in primates.

Inhibition of prostaglandin synthesis and its effect on uterine activity during established premature labor in sheep.

OBJECTIVE: Continuous infusion of the selective prostaglandin synthase type-2 inhibitor nimesulide, together with the oxytocin receptor antagonist atosiban, inhibits glucocorticoid induction of labor in sheep. We evaluated the effectiveness of this treatment commencing after the onset of premature labor when prostaglandin concentrations are already significantly elevated. METHODS: Premature labor was induced in chronically cannulated fetuses by constant fetal dexamethasone infusion. After the onset of active labor in each ewe, defined as uterine electromyographic (EMG) activity twice basal levels, ewes received combined nimesulide and atosiban (20.0 and 4.12 mg/kg per day, respectively; n = 6) or vehicle (n-methyl-2-pyrrolidone and saline each 1 mL/hour; n = 4) infusions for 48 hours. Maternal and fetal plasma PGFM (13,14-dihydro-15-keto PGF2alpha, the stable metabolite of prostaglandin (PG) F2alpha) and PGE2 concentrations were measured before, during, and after infusions. RESULTS: Four nimesulide- and atosiban-treated ewes successfully completed the 48-hour infusion period with no deliveries occurring during inhibitor treatment, or up to 6 hours after inhibitor treatment. Delivery was delayed in two other ewes, compared with control animals. Uterine EMG activity in nimesulide- and atosiban-treated ewes (n = 4) was significantly reduced during the 48-hour inhibitor treatment period. Maternal and fetal prostaglandin concentrations were significantly decreased in inhibitor-treated ewes during and after the infusions. CONCLUSIONS: The combination of nimesulide and atosiban treatment for 48 hours successfully inhibited the progression of active premature labor to delivery. This study further supports the potential value of this treatment regime for the inhibition of premature labor.

Effect of finasteride on behavioural arousal and somatosensory evoked potentials in fetal sheep.

This study examines the effect of inhibiting the synthesis of gamma-aminobutyric acid(A) (GABA(A)) agonist steroids on behavioural activity and somatosensory evoked potentials (SEP) in late gestation fetuses. Pregnane steroid production was suppressed by infusion of the 5alpha-reductase inhibitor, finasteride in chronically catheterised fetal sheep, 130-135 days gestation. Finasteride treatment (160 mg in 10 ml of vehicle over 2 h) significantly increased the incidence of fetal arousal during the period 4-10 h after commencing the infusion (P<0.05, n=6), whereas other behavioural parameters were not effected. In three of four animals, finasteride produced an increase in the amplitude of the N22 peak of the SEP during high voltage electrocortical activity. We conclude that suppression of pregnane steroid synthesis, by inhibition of the 5alpha-reductase enzyme, increases arousal activity in the fetus which is consistent with a reduction in GABA(A) receptor mediated inhibition.

Localization of p450scc and 5alpha-reductase type-2 in the cerebellum of fetal and newborn sheep.

Prenatally, neuroactive steroids that modulate GABAergic activity may be synthesized de novo within the fetal brain. We have examined changes in immunoreactivity staining for the steroidogenic enzymes cholesterol P450 side-chain cleavage (P450scc), and 5alpha-reductase type-2 in the cerebellum of late gestation (130-145 days gestation) fetal sheep and newborn lambs (1-4 weeks of age). Both enzymes were predominantly localized in the Purkinje cell body and dendrites of the fetal and newborn cerebellum, with weaker immunoreactivity in a few cells of the inner granular layer. P450scc immunoreactivity was present in Purkinje neurons expressing either of the neuronal microtubule associated proteins MAP1b/5 or MAP2a/b, but was absent from GFAP and HNK-1 positive cells. Soma of Purkinje neurons were also immunopositive for 5alpha-reductase type-2 in the fetuses, but expression decreased to just detectable levels in the 1-2 and 2-4 week old lambs. Both MAP1b/5- and MAP2a/b-positive Purkinje neurons showed 5alpha-reductase type-2 expression in the fetus, whereas the residual 5alpha-reductase staining in the newborn lamb was present only in MAP2a/b-positive Purkinje neurons. Allopregnanolone in the cerebellum decreased from 21.8+/-1.9 ng/g wet weight in fetuses at 140-145 days gestation to 6.7+/-0.5 ng/g in 2-4 week old lambs (P<0. 05). Thus, synthesis of neuroactive steroids from cholesterol is possible in cerebellar neurons in late gestation and persists into neonatal life, 5alpha-reductase type-2 expression is greater in the fetus compared to the neonate, and allopregnanolone concentrations in the cerebellum decrease significantly after birth.

Inhibition of premature labor in sheep by a combined treatment of nimesulide, a prostaglandin synthase type 2 inhibitor, and atosiban, an oxytocin receptor antagonist.

OBJECTIVE: The aim of this study was to compare the effects of the selective prostaglandin synthase type 2 inhibitor nimesulide, alone or in combination with the oxytocin receptor antagonist atosiban, on the progression of glucocorticoid-induced premature labor in sheep. Effects on circulating maternal and fetal prostaglandin concentrations and on fetal well-being were also examined. STUDY DESIGN: Premature labor was induced in ewes with long-term catheterized fetuses by infusion of dexamethasone (1 mg/d) starting at 138 +/- 1 days' gestation. Ewes also received an infusion of either nimesulide and atosiban (20.0 and 4.12 mg/kg per day, respectively; n = 5), nimesulide alone (20.0 mg/kg per day; n = 5), or vehicle only (n = 9). Plasma 13,14-dihydro-15-keto-prostaglandin F(2)(alpha) and prostaglandin E(2) concentrations were measured before and during infusions in plasma samples obtained from the maternal and fetal carotid arteries and the utero-ovarian vein. RESULTS: No fetuses from ewes treated with nimesulide and atosiban were delivered during treatment. These animals were killed electively 98.0 +/- 6.8 hours after the commencement of dexamethasone induction. This was significantly longer than the delivery times for those ewes treated with nimesulide alone (71.2 +/- 3.9 hours; n = 5) and for vehicle-treated ewes (51.4 +/- 1.7 hours; n = 9). Both maternal and fetal plasma 13, 14-dihydro-15-keto-prostaglandin F(2alpha) and prostaglandin E(2) concentrations in nimesulide and atosiban-treated ewes and in nimesulide-treated ewes decreased during treatment. In contrast, vehicle-treated ewes showed a significant increase in maternal and fetal plasma 13,14-dihydro-15-keto-prostaglandin F(2alpha) and prostaglandin E(2) concentrations during dexamethasone induction. Uterine electromyographic activity observed in nimesulide and atosiban-treated ewes was significantly suppressed with respect to activities in both vehicle- and nimesulide-treated ewes during the treatment period. All fetuses were alive at delivery or scheduled death. CONCLUSIONS: These results indicate that the combination of an inhibitor of prostaglandin endoperoxidase H synthase type 2 with an oxytocin receptor antagonist is more effective in inhibition of preterm labor than is treatment with a prostaglandin endoperoxidase H synthase type 2 inhibitor alone. The clinical use of atosiban to prevent the oxytocin-stimulated increase in uterine activity associated with labor in combination with nimesulide may permit reduction of the dose of nimesulide used to a level that has minimal impact on fetal well-being.

Characterisation of GABA(A) receptors in fetal, neonatal and adult ovine brain: region and age related changes and the effects of allopregnanolone.

Progesterone metabolites acting via GABA(A) receptors suppress central nervous system (CNS) activity. The aim of the present study was to examine binding characteristics of GABA(A) receptors in fetal, newborn and adult sheep brains using [(35)S]TBPS, and to determine the effects of allopregnanolone on this binding. Receptor affinity (K(D)) and density (B(MAX)) in the brainstem were not different in fetal, newborn (1-2 days old) and adult brains. In the hypothalamus K(D) and B(MAX) increased significantly in the fetus between 85 and 128 days gestation, and were then similar to postnatal and adult values. In the frontal cortex K(D) and B(MAX) increased progressively between 85 days and term ( approximately 147 days gestation), and were then not different from postnatal and adult values. The K(i) values for the GABA(A) receptor antagonist picrotoxin was similar at all ages. Allopregnanolone inhibited [(35)S]TBPS binding in the presence of 5 microM GABA, but enhanced binding in the absence of GABA. These results show that (i), functional GABA(A) receptors are present in the fetal brain from at least 85 days gestation; (ii), 3alpha-pregnane steroids modify receptor affinity in the late gestation fetal brain; and (iii) there are region-specific changes in GABA(A) receptor binding parameters. Steroid modulation of the GABA(A) receptor in the fetal brain is likely to influence fetal CNS activity in late gestation.

Effect of a neuroactive steroid infused into the cerebral ventricles of fetal sheep in utero using small infusion volumes.

Placental progesterone metabolites may influence fetal behaviour during late gestation. We have investigated the effect of the neuroactive metabolite, pregnanolone, on the sleep/awake behaviour of fetal sheep. These studies can only be effectively performed in unanesthetized animals with chronically implanted leads and catheters. The relatively large internal volumes of these catheters raises problems for cerebroventriclear infusions. Therefore, we developed a device with negligible deadspace ( approximately 10 microl) consisting of a ventricular cannula attached to a small cap which allowed the instillation of microl volumes of neuroactive steroids into a cerebral ventricle of fetal sheep in utero. Two catheters attached to the cap allow it to be filled in a push-pull configuration. The smaller internal diameter of the ventricular cannular prevents the solution entering the ventricle until the outflow catheter is occluded and solution is forced through the probe. Using this device, the infusion of 5beta-pregnane-3alpha-ol-20-one (pregnanolone, 1 mg), in 35% hydroxypropyl-beta-cyclodextrin, resulted in a marked suppression of fetal behaviour parameters indicative of arousal, while vehicle alone did not affect these parameters. We conclude that neuroactive steroids can be infused into the cerebroventricular system of chronically catheterised fetal sheep in small volumes and that pregnanolone may act directly to influence fetal behaviour.

Efficacy of the selective prostaglandin synthase type 2 inhibitor nimesulide in blocking basal prostaglandin production and delaying glucocorticoid-induced premature labor in sheep.

OBJECTIVE: The purpose of the study was to determine the effects of selective prostaglandin synthase type 2 inhibitors on basal prostaglandin concentrations in the fetal and maternal circulations and on the labor-associated increase in prostaglandin production in sheep. STUDY DESIGN: The effects of maternal nimesulide (0.01, 0.1, and 1 mg/kg) and 6-methoxy-2-naphthylacetic acid (1, 5, and 10 mg/kg) administration were examined (n = 5) at 134 +/- 1 days' gestation. At 138 days' gestation premature labor was induced by fetal dexamethasone infusion (1 mg/d). Ewes were treated with either vehicle or nimesulide infusion (20 mg. d-1. kg-1, n = 5 per group). RESULTS: Nimesulide and 6-methoxy-2-naphthylacetic acid decreased basal prostaglandin production in a concentration-dependent manner. Delivery of nimesulide-treated ewes was delayed by >/=17 hours with respect to that of control ewes (53.9 +/- 2.6 hours). In 2 nimesulide-treated ewes labor did not progress to delivery despite membrane rupture. The increase in prostaglandin concentrations usually seen during dexamethasone-induced labor was abolished in nimesulide-treated ewes and also in their fetuses. CONCLUSIONS: Highly selective inhibitors of prostaglandin endoperoxidase H synthase 2 may be required to spare fetal prostaglandin production and limit potential side effects during the suppression of preterm labor.

Effects of pregnanolone on behavioural parameters and the responses to GABA(A) receptor antagonists in the late gestation fetal sheep.

Placental progesterone metabolites may suppress fetal behaviour by interacting with GABA(A) receptors. In an initial study, the effect of 5beta-pregnan-3alpha-ol-20-one (pregnanolone) given as a bolus (2.5 and 5.0 mg) or infused at a rate of 25 mg/h was investigated in unanaesthetized, catheterized fetal sheep, 127-135 days gestation. The incidence of fetal breathing movements (FBM) and behavioural arousal activity, defined as nuchal muscle electromyographic (EMG) activity during low voltage electrocortical (LV ECoG) activity were suppressed by pregnanolone administered as a bolus, while the pregnanolone infusion produced a significant decrease in arousal and EOG activity, and an increase in the presence of HV ECoG. The effect of pregnanolone on fetal behaviour and arousal induced by the GABA(A) antagonist picrotoxin was also investigated. Picrotoxin was given as a bolus (approximately 300 microg/kg) and pregnanolone was subsequently administered as a bolus (5.0 mg), and behavioural parameters were recorded and analysed. The incidence of arousal and FBM were 1.1 +/- 1.6 min/10 min and 2.5 +/- 2.3 min/10 min, respectively, before picrotoxin treatment and increased during the 10-20 and 20-30 min epochs after picrotoxin treatment (arousal: 5.0 +/- 2.2 and 6.5 +/- 3.6 min/10 min, respectively, n = 6, P < 0.05; FBM: 7.3 +/- 3.2 and 9.3 +/- 1.2 min/10 min, respectively, n = 6, P < 0.05). The picrotoxin-induced increases in arousal and FBM were significantly suppressed (n = 6, P < 0.05) by pregnanolone treatment to 1.6 +/- 1.5 min/10 min and 4.6 +/- 2.3 min/10 min, respectively. We conclude that; (i) the GABA(A) active steroid pregnanolone suppresses basal and picrotoxin-induced fetal arousal and FBM; and (ii) steroid sensitive GABA(A) receptors may regulate fetal behaviour and breathing.

Effect of pregnane steroids on electrocortical activity and somatosensory evoked potentials in fetal sheep.

The effect of infusing the neuroactive steroids pregnanolone and iso-pregnanolone on somatosensory evoked potentials (SEP) and electrocortical (ECoG) activity was studied in unanaesthetised fetal sheep, 130-135 days gestation. Intravenous infusion of pregnanolone (6 mg/kg per h) significantly increased the proportion of high voltage ECoG (56.1+/-4.8% vs. control 43.5+/-3.2%, P < 0.05), and decreased low voltage ECoG (43.9+/-4.8% vs. control 56.6+/-3.2%, P < 0.05). Pregnanolone treatment decreased the amplitude of the N25 peak of the SEP (89.9+/-2.8% of control, P < 0.05) evoked following stimulation of the skin of the upper lip. In contrast, iso-pregnanolone treatment had no effect on ECoG activities, or on the amplitude and latency of peaks in the SEP. We conclude that 3alpha-hydroxy pregnane steroids are active at GABA(A) receptors in fetal sheep and can modulate sleep/wake activity before birth.