Dual specificity phosphatase 9 (DUSP9) expression is down-regulated in the severe pre-eclamptic placenta.

OBJECTIVES: Intrauterine growth restriction (IUGR) and pre-eclampsia are severe and clinically important manifestations of placental insufficiency. In the mouse, dual specificity phosphatase 9 (DUSP9) is critical to the normal development of the placenta, where knock-outs are growth restricted and have a placental phenotype similar to that seen in syndromes of human placental insufficiency. Our purpose was to characterize DUSP9 expression in normal human pregnancy and in cases of placental insufficiency. STUDY DESIGN: We used RT-PCR, immuno-histochemistry and Western blotting to characterize DUSP9 gene expression and protein levels across human gestation and in pregnancies complicated by severe IUGR and/or severe pre-eclampsia. DUSP9 promoter methylation was studied in pathologic and pre-term control placentas to investigate potential epigenetic regulation. First trimester villous explants and BeWo cells were treated with DUSP9 silencing RNA to determine the effect on downstream pathways. Placental hypoxia is a hallmark of pre-eclampsia; therefore explants were subjected to hypoxic culture conditions to determine the effect of oxygen on DUSP9 expression in vitro. RESULTS: DUSP9 expression was evident in villous trophoblast and declined during development. DUSP9 protein was significantly lower in severe pre-eclamptic placentas compared to severe growth restriction. This was not epigenetically mediated by promoter hyper-methylation, and the downstream pathway ERK1/2 was not significantly affected. DUSP9 expression in first trimester explants was significantly decreased by 74 ± 20% in hypoxic (3% oxygen) culture conditions. In BeWo cells and explanted placental villi treated with DUSP9 silencing RNA, expression of DUSP9 was down-regulated by 61% and 62% respectively. There was a trend to increased phosphorylation of the downstream target ERK1/2 in DUSP9 down-regulated BeWo cells and explanted placental villi. CONCLUSION: DUSP9 protein levels were markedly suppressed in severe pre-eclampsia, but not in severe IUGR. This suppression might be attributable to the prolonged hypoxic conditions found in pre-eclampsia.

A distinct microvascular endothelial gene expression profile in severe IUGR placentas.

The placental microvasculature is essential for efficient transfer of gases, nutrients and waste between the mother and fetus. Microvascular hypoplasia of the terminal villi is a common pathology in severe Intra Uterine Growth Restriction (IUGR). We used novel methods to obtain placental micro-vascular endothelial cells (PlMEC) from preterm control placentas (n = 3) and placentas from pregnancies with severe IUGR (n = 6) with absent or reversed end-diastolic velocity in the umbilical artery. Distal placental villous tissue was collected to enrich for intermediate and terminal villi. Tissue was digested and PlMEC positively selected using tocosylated magnetic Dynabeads labeled with Human Endothelial Antigen lectin. The purity of the PlMEC (94 ± 2 SD %) was assessed by CD31 and vimentin immunocytochemistry. RNA was extracted from the PlMEC samples and subjected to Affymetrix microarray analysis (U133Plus2 array chips). Comparison of preterm and IUGR PlMEC gene expression profiles identified BTNL9 and NTRK2 transcripts to be upregulated and SAA1 and SLAMF1 transcripts to be downregulated in all 6 IUGR cases relative to preterm controls. A third downregulated gene GNAS was identified to be near significance. Changes were demonstrated to be significant at the mRNA level by Real Time PCR in the PlMEC samples. Changes in the IUGR endothelium were confirmed at the protein level by immunohistochemistry for the 3 with available antibodies. We used a tissue microarray constructed from an independent cohort of placental samples from severe IUGR (n = 7), preeclamptic (n = 7), preterm control (n = 6) and term control (n = 6) pregnancies. Results confirmed differential endothelial expression of BTNL9, NTRK2 and SLAMF1 in IUGR versus preterm and term samples. These studies are the first to characterize PlMEC gene expression profiles thus we have advanced our understanding of the molecular basis of placental micro-vascular pathophysiology in fetal growth restriction.

The missense mutation W290R in Fgfr2 causes developmental defects from aberrant IIIb and IIIc signaling.

Missense mutations in the Fibroblast Growth Factor Receptor 2 (FGFR2) have been identified in human craniosynostotic syndromes such as Crouzon (CS) and Pfeiffer (PS). FGFR2 has two major isoforms, IIIb and IIIc, generated through alternative splicing with their own temporal, spatial, and ligand-binding specificities. In this study, we report the identification and characterization of a missense mutation in codon 290 of murine Fgfr2 (W290R). The defects in W290R mutants are suggestive of disruption of signalling in both IIIb and IIIc isoforms of the Fgfr2 gene. Heterozygous mutants presented with features resembling those found in patients with CS. Fgfr2(W290R) homozygotes displayed constitutive FGFR2 activation with increased, but correct tissue-specific, expression of the IIIb and IIIc isoforms in many of the defective organs. Our Fgfr2(W290R) mouse model thus represents an excellent mouse model of CS to probe the many questions around the pathogenesis of craniosynostotic birth defects consequent to defects in FGF signaling.

3D visualisation and quantification by microcomputed tomography of late gestational changes in the arterial and venous feto-placental vasculature of the mouse.

This study evaluates microcomputed tomography (micro-CT) as a method to obtain quantitative three-dimensional (3D) information on the arterial and venous vasculature of the mouse placenta. Surface renderings at embryonic days (E) 13.5, 15.5, and 18.5 (full term) revealed that the arterial and venous vasculature branched within the chorionic plate whereas only the arterial vasculature deeply penetrated the placenta. Umbilical vessel diameters measured by micro-CT did not significantly differ from those measured non-invasively in vivo by ultrasound biomicroscopy. Variability in umbilical diameters, and surface area and volume measurements of arterial and venous vascular trees due to experimental error was low relative to biological variability, and significant inter-litter differences within gestational ages were detected. Furthermore, umbilical vessel diameter increased significantly and incrementally to an arterial diameter of 0.631+/-0.009 mm and a venous diameter of 0.690+/-0.018 mm at E18.5. Umbilical vein diameter was 3-9% greater than the artery, and both were significantly correlated with embryonic body weight (R> or =0.96). Surface area and volume were determined for vessels greater than the minimum resolvable diameter of 0.03 mm which therefore excluded capillaries. Arterial surface area and volume were unchanged from E13.5-15.5 but then more than doubled at E18.5 (to 170+/-13 mm(2) and 7.2+/-0.8mm(3), respectively). Venous surface areas and volumes changed similarly with development although surface areas were lower than their arterial counterparts. We conclude that micro-CT has sufficient accuracy and precision to quantify late gestational changes in the 3D structure of the arterial and venous vasculature of the mouse placenta.

Ultrasonic detection and developmental changes in calcification of the placenta during normal pregnancy in mice.

High resolution ultrasound imaging of the mouse placenta during development revealed highly echogenic foci localized near the materno-placental interface in early gestation and, near term, in the placental labyrinth (the exchange region of the placenta). Echogenic foci and calcium deposits identified in histological sections using Alizarin red staining showed similar localization and changes with gestation. Calcium deposits caused the echogenic foci because incubating uteri in a decalcifying solution eliminated both the deposits and echogenic foci. Transmission electron microscopy, X-ray microanalysis, and electron diffraction were used to show that deposits were calcium hydroxyapatite crystals. Calcium deposits were extensive and densely packed at days 7.5-9.5 of gestation at the border between the maternal decidua and the fetal trophoblast giant cells of ectoplacental cone. After the formation of the chorio-allantoic placenta (approximately day 10.5), calcification deposits appeared larger and more rarefied but were still localized at the border between the maternal decidua and the fetal trophoblast giant cells of the placenta. Calcification deposits were not observed in the labyrinthine region of the mouse placenta until > or = day 15.5 (day 18.5 is full term). We conclude that deposits of calcium hydroxyapatite crystals in the mouse placenta are detectable by high resolution ultrasound imaging. These deposits provide an ultrasound detectable marker of the maternal-placental interface that is particularly prominent during the establishment of the chorio-allantoic placenta between days 7.5 and 9.5 of gestation.

A new ultrasound instrument for in vivo microimaging of mice.

We report here on the design and evaluation of the first high-frequency ultrasound (US) imaging system specifically designed for microimaging of the mouse. High-frequency US or US biomicroscopy (UBM) has the advantage of low cost, rapid imaging speed, portability and high resolution. In combination with the ability to provide functional information on blood flow, UBM provides a powerful method for the investigation of development and disease models. The new UBM imaging system is demonstrated for mouse development from day 5.5 of embryogenesis through to the adult mouse. At a frequency of 40 MHz, the resolution voxel of the new mouse scanner measures 57 microm x 57 microm x 40 microm. Duplex Doppler provides blood velocity sensitivity to the mm per s range, consistent with flow in the microcirculation, and can readily detect blood flow in the embryonic mouse heart, aorta, liver and placenta. Noninvasive UBM assessment of development shows striking similarity to invasive atlases of mouse anatomy. The most detailed noninvasive in vivo images of mouse embryonic development achieved using any imaging method are presented.

Applications for multifrequency ultrasound biomicroscopy in mice from implantation to adulthood.

A new multifrequency (19-55 MHz) ultrasound biomicroscope with two-dimensional imaging and integrated Doppler ultrasound was evaluated using phantoms and isoflurane-anesthetized mice. Phantoms revealed the biomicroscope's lateral resolution was between 50 and 100 microm, whereas that of a conventional 13 MHz ultrasound system was 200-500 microm. This difference was apparent in the markedly higher resolution images achieved using the biomicroscope in vivo. Transcutaneous images of embryos in pregnant mice from approximately 2 days after implantation (7 days gestation) to near term (17.5 days) were obtained using frequencies from 25 to 40 MHz. The ectoplacental cone and early embryonic cavities were visible as were the placenta and embryonic organs throughout development to term. We also evaluated the ability of the biomicroscope to detect important features of heart development by examining embryos from 8.5 to 17.5 day gestation in exteriorized uteri using 55 MHz ultrasound. Cardiac looping, division of the outflow tract, and ventricular septation were visible. In postnatal imaging, we observed the heart and kidney of neonatal mice at 55 MHz, the carotid artery in juveniles (approximately 8 g body wt) and adults (approximately 25 g body wt) at 40 MHz, and the adult heart, aorta, and kidney at 19 MHz. The coefficient of variation of carotid and aortic diameter measurements was 1-3%. In addition, blisters in GRIP1 -/- embryos and aortic valvular stenosis in two adults were readily visualized. Using image-guided Doppler function, low blood velocities in vessels as small as 100 microm in diameter including the primitive heart tube at day 8.5 were measurable, but high blood velocities (>37.5 cm/s) such as in the heart and large arteries in late gestation and postnatal life were off-scale. Accurate cardiac dimension measurements were impeded by poor temporal resolution (4 frames/s). In summary, the multifrequency ultrasound biomicroscope is a versatile tool well suited to detailed study of the morphology of various organ systems throughout development in mice and for hemodynamic measurements in the low velocity range.

Trophoblast functions, angiogenesis and remodeling of the maternal vasculature in the placenta.

One of the most important local adaptations to pregnancy is the change in maternal blood flow to the implantation site. In rodents and primates, new blood vessels form through angiogenesis, dilate and then become modified such that the blood enters into trophoblast cell-lined sinuses (hemochorial). Evidence from gene knockout mice suggests that factors from the placenta regulate the uterine vasculature. Consistent with this, trophoblast giant cells produce a number of angiogenic and vasoactive substances that may mediate these effects. Teratocarcinomas containing large numbers of trophoblast giant cells (derived from Parp1 gene-deficient ES cells) show similar 'hemochorial' host blood flow, implying that the effects are not specific to the uterine vascular bed. As in primates, murine trophoblast cells also invade into the uterine arteries of the mother. However, in normal pregnancy, dilation of the uterine arteries may be largely mediated by the effect of uterine natural killer cells.

TGF-beta 3 expression during umbilical cord development and its alteration in pre-eclampsia.

Members of the TGF-beta family have been shown to play an important role in numerous tissues during development. In the present study we have investigated the spatial and temporal expression of TGF-beta 3, in human umbilical cord development. Total TGF-beta 3 protein content, assessed by immunoblotting, increased with advancing gestation as did immunostaining and mRNA in Wharton's jelly fibroblasts. Immunohistochemical analysis revealed that TGF-beta 3 was present in all cell types. Temporal changes in TGF-beta 3 expression were observed in the vascular smooth muscle cells, such that with advancing gestation TGF-beta 3 protein expression and became mostly restricted to the extracellular compartment of the vascular media. This was associated with a decrease in TGF-beta 3 mRNA expression in umbilical vascular smooth muscle cells. Of clinical significance, umbilical cords from pregnancies complicated by pre-eclampsia, showed a significant reduction in total TGF-beta 3 protein expression when compared to those of age-matched patients. Both TGF-beta 3 mRNA and protein expression were downregulated in the endothelium and smooth muscle layers of the umbilical arteries, as well as in the Wharton jelly fibroblasts. Our data demonstrate that during umbilical cord development TGF-beta 3 expression is spatially and temporally regulated and that TGF-beta 3 expression is altered in umbilical cords of pregnancies complicated by pre-eclampsia. We speculate that the downregulation of TGF-beta 3 expression found in pre-eclamptic umbilical cord may contribute to the abnormal structure and mechanical properties seen in these pathological umbilical cords.

Perinatal changes in choroidal 15-hydroxyprostaglandin dehydrogenase: implications for prostaglandin removal from brain.

We have previously shown in the sheep fetus at 0.7 and 0.9 gestation that the choroid plexus, unlike brain parenchyma, catabolizes prostaglandins (PGs). Peculiarly, in the choroid plexus, PGE(2) catabolism persists throughout the neonatal period to abate in the adult, while PGF(2alpha) catabolism abates shortly after birth. To explain this differential behavior and elucidate the function of catabolic enzymes, we examined the cellular location and activity of the rate-limiting enzyme for PGE(2) and PGF(2alpha) catabolism, 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Immunofluorescence histochemistry and immunogold electronmicroscopy revealed abundant 15-PGDH expression in the epithelial cytosol close to the brush-border membrane at 0.7 and 0.9 gestation. In contrast, at 5 and 15 days postnatal, 15-PGDH was found throughout the cytosol of stromal fibroblasts. No staining was observed at either location in pregnant adults. PGF(2alpha) catabolism was minimal in the total homogenate and 100000xg supernatant of the fetal choroid plexus at 0.7 and 0.9 gestation, while PGE(2) catabolism was evident at 0.7 gestation only. In contrast, both PGs were catabolized in minced specimens at either age. In conclusion, our study shows immunoreactive 15-PGDH in the choroid plexus from fetal and neonatal, but not pregnant adult, sheep. Results suggest that PGE(2) catabolism is not as critically dependent as that of PGF(2alpha) on tissue integrity and 15-PGDH location. Given the key role being assigned to the choroid plexus in PG removal from brain, we speculate that persistence of PGE(2) catabolism into the early postnatal period protects against central respiratory depression caused by the compound during this susceptible stage of development.

Developmental changes in respiratory, febrile, and cardiovascular responses to PGE(2) in newborn lambs.

PGE(2) has centrally mediated respiratory, febrile, and cardiovascular effects that markedly differ between fetal and adult life. We hypothesized that the transition from fetal to adult responses to PGE(2) occurs in the newborn period. Thus effects of an intracarotid infusion of PGE(2) (3 microg/min for 60 min) were determined in unanesthetized newborn lambs at 5, 10, and 15 days after birth. At 5 days, PGE(2) reduced central CO(2) sensitivity, reduced lung ventilation due to a decrease in breathing frequency, and induced hypercapnia. By 15 days, these effects of PGE(2) had waned significantly. In contrast, phasic (expiratory) thyroarytenoid muscle electromyogram activity, number of short apneas, and incidence of Biot periodic breathing were similarly increased at all three ages. PGE(2) induced a sustained fever at 10 and 15 days. Heart rate and mean arterial blood pressure were unchanged in contrast to marked increases observed by others in adults. Results showed that the transition from fetal to adult respiratory and febrile responses to PGE(2) occurs in early postnatal life, whereas adult cardiovascular responses develop later in life in sheep.

Glucose metabolism is elevated and vascular resistance and maternofetal transfer is normal in perfused placental cotyledons from severely growth-restricted fetuses.

We hypothesized that placental resistance was elevated and transfer reduced in cotyledons from intrauterine growth-restricted (IUGR) fetuses. We perfused 10 cotyledons from term, normally grown fetuses, six from preterm, normally grown fetuses with normal umbilical arterial end-diastolic velocities (EDV), and six from preterm IUGR fetuses (<3rd centile) with absent or reversed umbilical arterial EDV. Perfused cotyledons were pressure-fixed, and villi were observed by scanning electron microscopy. The groups did not differ in fetoplacental resistance at baseline; neither did they differ in the change in resistance that followed the administration of nitroglycerin or angiotensin II. The increase in resistance during hypoxia was similar in the two preterm groups but greater in the term than in the preterm normally grown group (p < 0.05). Groups did not differ in net maternofetal transfer of oxygen or glucose, or in clearance of aminoisobutyric acid or antipyrine. However, glucose consumption was doubled in cotyledons of preterm IUGR versus preterm normally grown fetuses (p < 0.05). Terminal villi of perfused cotyledons from preterm IUGR fetuses displayed less terminal villous branching and budding than preterm controls, as anticipated from previous work. IUGR fetuses with absent or reversed umbilical arterial EDV in vivo may have high placental resistance due to a vasoconstrictive rather than anatomic abnormality and an elevated placental glucose consumption that may impair glucose transfer.

Differential uptake and catabolism of prostaglandin (PG)E(2) versus PGF(2alpha) in the sheep choroid plexus during development.

The early postnatal decrease in prostaglandin (PG)E(2) levels in cerebrospinal fluid (CSF) likely contributes to the establishment of continuous breathing. To elucidate mechanisms underlying this event, choroid plexuses from lateral (L-CP) and third/fourth (III/IV-CP) ventricles were incubated with [3H]-PGE(2) and label uptake (tissue-to-medium ratio for radioactivity, T/M) and catabolism (%radioactivity associated with metabolites, PGM) were measured. [3H]-PGF(2alpha) was a reference. Uptake of [3H]-PGE(2) was lower than [3H]-PGF(2alpha) in the term fetus (L-CP: 5.9+/-0.5 vs. 9.6+/-0. 9, n=11; III/IV-CP: 2.7+/-0.4 vs. 7.7+/-1.0, n=5) and 17 d lamb (L-CP: 5.3+/-0.8 vs. 11.0+/-1.2, n=7; III/IV-CP: 3.1+/-0.2 vs. 11. 6+/-2.8, n=3 and 4, respectively). This difference was not significant in the pregnant adult. Release of the two compounds was similar and did not change with age. [3H]-PGE(2) uptake was reduced by probenecid (1 mM) and excess PG (60 microM PGE(2) or PGF(2alpha)). Excess PG also reduced catabolism in the fetus, which was extensive for [3H]-PGE(2) and [3H]-PGF(2alpha)60%). In the lamb, catabolism remained high for [3H]-PGE(2) (L-CP: 64+/-4%, n=7; III/IV-CP: 41+/-4%, n=3), but not [3H]-PGF(2alpha) (L-CP: 26+/-4%, n=7; III/IV-CP: 4+/-1%, n=4). In the pregnant adult, catabolism was above background only for [3H]-PGE(2) in the L-CP (26+/-5%, n=11). Unlike the perinatal animal, this catabolism was reduced by probenecid. In conclusion, PGE(2) uptake and catabolism operate independently in the choroid plexus from perinatal sheep. Differences between PGE(2) and PGF(2alpha) are developmentally-regulated for both mechanisms. While neither process explains the postnatal decrease in CSF PGE(2), both may help keep CSF levels low during early postnatal development.

Determinants of mechanical properties in the developing ovine thoracic aorta.

We previously reported changes in mechanical properties and collagen cross-linking of the ovine thoracic aorta during perinatal development and postnatal maturation, and we now report changes in biochemical composition (elastin, collagen, and DNA contents per mg wet wt) over the same developmental intervals. A comparison of results from the present and previous studies has yielded novel and important observations concerning the relationship between aortic mechanics and composition during maturation. Developmental changes in aortic incremental elastic modulus at low tensile stress (E(low)) closely followed changes in relative elastin content (i.e., per mg wet wt). An 89% increase in E(low) during the perinatal period was associated with a 69% increase in relative elastin content, whereas neither variable changed during postnatal life. Incremental elastic modulus at high tensile stress (E(high)) did not change during the perinatal period but increased 88% during postnatal life. This pattern closely paralleled changes in collagen cross-linking index, which did not change perinatally but almost doubled postnatally. In contrast, relative collagen content (per mg wet wt) increased only slightly from fetal to adult life, a trend that was unrelated to aortic mechanics. Substantial, progressive decreases in measures of wall viscosity (pressure wave attenuation coefficient and viscoelastic phase angle) from fetal to adult life followed the pattern observed for relative DNA (smooth muscle cell) content (per mg wet wt). Our findings suggest that accumulation of elastin per milligram wet weight contributes most to developmental changes in E(low), change in collagen cross-linking is the primary determinant of developmental changes in E(high), and cell accumulation contributes most to developmental changes in wall viscosity.

Arterial pressure, vascular input impedance, and resistance as determinants of pulsatile blood flow in the umbilical artery.

The flow pulsatility index, the ratio of flow pulse amplitude to mean flow over the cardiac cycle, has been used to quantify pulsatility of blood flow in the umbilical artery. In experiments with fetal sheep, we showed that the flow pulsatility index in the umbilical artery is accurately estimated by the ratio of total umbilico-placental vascular resistance (mean arterial pressure divided by mean umbilical flow) divided by fundamental impedance (umbilical vascular impedance at the heart rate frequency) times the pulsatility index (pulse/mean) of the arterial pressure that drives flow through this bed. The pulsatility index of arterial pressure is primarily determined by upstream factors (e.g. heart rate) whereas fundamental impedance depends primarily on the radius and viscoelastic wall properties of the umbilical artery. An increase in resistance in the microcirculation and/or veins causes proportional changes in the flow pulsatility index because these sites have little influence on fundamental impedance. However, an increase in resistance in the highly vasoactive umbilical arteries has offsetting effects on impedance and resistance; consequently, flow pulsatility changes little even when arterial vasoconstriction markedly reduces mean flow. We conclude that when arterial pressure pulsatility is stable, a change in the flow pulsatility index provides a useful indication of a change in resistance in the microcirculation and/or veins but will not reliably detect a resistance change in the artery.

Atrophic remodeling of the artery-cuffed artery.

Increased arterial wall tension stimulates growth and remodeling of arteries, but little is known about the effects of decreased wall tension, despite its developmental and pathological significance. Consequently, we cuffed 1 carotid artery in rabbits with a portion of the contralateral artery to off-load circumferential wall tension. The model produced rapid and extensive atrophy of the cuffed artery that yielded decreases in the DNA content of the cuffed artery (a measure of cell number) from 8.0+/-0.5 microgram/cm of in situ vessel length to 5.6+/-0.5 microgram/cm at 21 days postoperatively. The elastin content of the cuffed artery was also significantly reduced, from 399+/-17 to 283+/-17 microgram/cm, and collagen content was reduced from 468.0+/-59.0 to 154+/-24 microgram/cm (P<0.05) at 21 days postoperatively. Detection of DNA oligonucleosomes by gel electrophoresis implicated apoptotic cell death in remodeling due to cuffing. Upregulation of matrix metalloproteinases (MMPs), including MMP-2, MMP-9, and unidentified gelatinases, indicated that these enzymes may also be involved in remodeling. No further changes in wall structure were seen between 3 weeks and 6 months, and the excised artery that was used as a cuff exhibited normal medial morphology for at least 6 months postoperatively. We infer from these experiments that off-loading of arterial wall tension induces rapid and extensive atrophy of the arterial media.

Interconversion of cortisol and cortisone by 11beta-hydroxysteroid dehydrogenases type 1 and 2 in the perfused human placenta.

The human placenta contains two types of 11beta-hydroxysteroid dehydrogenase (11beta-HSD). The exclusive oxidase 11beta-HSD2 has been suggested to protect the fetus from high levels of maternal glucocorticoids by converting cortisol to inactive cortisone. Perfused term human placenta was used to examine the activity of the oxoreductase 11beta-HSD1 and to determine the regulation of cortisol effects on placental vascular tone and corticotropin-releasing hormone (CRH) output by 11beta-HSD. Radioimmunoassay showed that there was substantial cortisol (295+/-57 nM) detected in the fetal vein upon perfusion of cortisol (2 microM; perfusion rate, 12 ml/min) into the maternal intervillous space. Output of cortisol increased to 559+/-22 nM on the fetal side (P<0.05) with concurrent perfusion of carbenoxolone (CBX; 1 microM), a non-specific 11beta-HSD inhibitor. Cortisol formation increased in a dose-dependent manner with infusion of cortisone (0.1-2 microM) into the maternal intervillous space reaching 15 and 23 nM in fetal and maternal venous outflows respectively at 2 microM cortisone perfusion. There was no significant effect of cortisol either alone or in combination with CBX on the fetal arterial perfusion pressure, but cortisol perfusion increased CRH output into the fetal vein. It is concluded that activities of both 11beta-HSD1 and -2 are demonstrable in perfused human placenta in vitro, and these enzymes affect transplacental glucocorticoid transfer. These activities may provide a precise mechanism to control the passage of maternal glucocorticoids to the fetal circulation, and to regulate glucocorticoid effects within the placenta.

Cardiovascular responses attenuate with repeated NO synthesis inhibition in conscious fetal sheep.

The cardiovascular effects of repeated administration of the nitric oxide (NO) synthesis inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) were assessed daily for 3 days in fetal sheep near term (124-126 days gestation) beginning 4 days after surgery (n = 7). In the first hour on day 1, fetal infusion of L-NAME (30 mg bolus, 6 mg/min infusion iv for 3 h) significantly increased fetal arterial pressure from 41 +/- 2 to 58 +/- 3 mmHg, decreased heart rate from 173 +/- 5 to 134 +/- 3 beats/min, increased umbilicoplacental resistance from 0.16 +/- 0.02 to 0.28 +/- 0.07 mmHg.ml-1.min, and inhibited the hypotensive response to acetylcholine (ACh; 2 micrograms iv bolus). All changes were sustained except for arterial pressure, which decreased significantly to 50 +/- 3 mmHg in the third hour. Within 17 h, all cardiovascular variables returned to control. L-NAME readministered on days 2 and 3 had no effect on cardiovascular variables. L-NAME did not potentiate the pressor response to angiotensin II on day 2 and caused a surprising attenuation of the pressor response to endothelin-1 on day 3. We conclude that, whereas NO normally contributes to low arterial pressure, high heart rate, and low umbilicoplacental vascular resistance in fetal sheep near term, the role of NO in these functions is replaced by an alternate mechanism within 17 h after NO synthesis inhibition with L-NAME.