Helen H. Kay

Antenatal Steroid Treatment and Adverse Fetal Effects: What Is the Evidence?

This article reviews current animal and human data regarding possible adverse fetal effects from antenatal steroid treatment. Although it is now well accetped that such treatment is of benefit to fetal lung development, the potential for adverse fetal outcomes as a result of single or multiple glucocorticoid dosing has not been widely recognized. There are now growing concerns, based on animal and some human data, that repeated antenatal doses could lead to a decrease in birth weight, a decrease in fetal brain and other organ size, and abnormal neuronal development. Previous investigations have been hampered by nonstandardization in the type of glucocorticoid, route of delivery, timing of administration, and number of treatment courses. It is recommended that these concerns be addressed through large randomized, controlled clinical trials. In the meantime, it would be prudent to minimize antenatal steroid treatments to a single course with repeated dosing only if there is a persistent threat of preterm delivery. THe practice of giving weekly injections of steroids starting at fetal viability and continuing into the third trimester is not supported.

DECREASED LEVELS OF POLYUNSATURATED FATTY ACIDS IN PREECLAMPSIA

Plasma levels of polyunsaturated fatty acids in the n-3 and n-6 classes, which include linoleic, linolenic, arachidonic, eicosapentaenoic, and docosahexaenoic acids, were quantified with high-performance liquid chromatography in nonpregnant volunteers and in patients with normal pregnancies or preeclampsia at term. The total polyunsaturated fatty acid levels were not significantly different between nonpregnant and normal pregnant patients but was significantly lower in the preeclamptic patients compared with normal pregnant patients. This decreased level could represent altered fatty acid metabolism or altered storage and mobilization from lipid pools. Compared with nonpregnant patients, normal pregnant patients had significantly higher levels of eicosapentaenoic and docosahexaenoic acid. This may reflect normal physiologic changes in pregnancy, and the decreased level of eicosapentaenoic acid seen in preeclamptic patients may play a significant role in the pathophysiology of preeclampsia.

cDNA microarray analysis of gene expression profiles in human placenta: up-regulation of the transcript encoding muscle subunit of glycogen phosphorylase in preeclampsia.

Objective: Third-trimester human placentas from normal and preeclamptic pregnancies were evaluated for possible changes in gene expression patterns by microarray analysis. Methods: Placentas from four normal pregnancies and four pregnancies complicated by preeclampsia were studied. In a preliminary effort to identify possible differences between the two groups, complementary DNA (cDNA) probes were prepared from pooled total RNA by reverse transcription in the presence of 33-P-dCTP. After hybridization to human GeneFilter cDNA microarrays (GF211; Research Genetics, Huntsville, AL), 319 positive signals were detected above background out of a possible 4131 human cDNAs spotted on the filters. Results: Ten most highly expressed mRNA species, ten most up-regulated, and ten most down-regulated genes in placentas from both groups of patients were identified for future studies. Of the 319 positive hybridizations, one transcript was clearly evaluated in preeclamptic pregnancy. This cDNA encodes the muscle subtype of glycogen phosphorylase (GP-M) and was increased more than 2.8-fold (P < .05) in the preeclamptic placentas. In contrast, cDNA for glycogen synthase (muscle and liver isoforms) was not significantly increased, being near the limits of detection. The preeclampsia-induced increase of placental GP-M mRNA expression (approximately 3.5-fold) was confirmed by northern blot analysis. Conclusion: We conclude that microarray analysis can detect trends in mRNA and gene expression in placentas from normal and preeclamptic pregnancies and may be further studied in a more targeted fashion. We found that placental GP-M mRNA level is up-regulated in preeclampsia, which is consistent with previous reports of increased glycogen phosphorylase activity, and we suggest that it may be largely regulated at the level of transcription. Further studies may determine whether such up-regulation might be a response to hypoxia.

Markers of oxidative stress in placental villi exposed to ethanol.

Objective: Ethanol exposure during pregnancy may result in fetal alcohol syndrome (FAS). The mechanism by which this occurs is unknown. Recent studies in several organ systems, including the placenta, suggest that oxidative stress is involved. In this study we investigated the presence and levels of three oxidative stress markers in placental villous tissue exposed to ethanol. Methods: Villous tissues from normal placentas were perfused with Dulbecos modified Eagles medium (DMEM) with HEPES buffer, sodium bicarbonate, and glucose at pH 7.4. After stabilization, 100 mM ethanol was added to the perfusate. After 2 hours of perfusion, the tissue was removed, fixed and stained for nitrotyrosine, 4-hydroxy-2-nonenal (4HNE) and 8-hydroxyguanosine (8-OHDG). Staining within the trophoblasts was quantified with densitometry. Results: Nitrotyrosine and 4HNE immunostaining was seen in the trophoblasts. 4HNE was also seen in the stroma. In contrast, 8-OHDG was seen only in the stroma and endothelial cells in the fetal circulation. Ethanol exposure significantly increased nitrotyrosine levels in the trophoblasts beyond levels in the control tissue. Nitrotyrosine and 8-OHDG levels were also increased in stroma. Conclusion: Within the placental villi, markers of oxidative stress are present in the trophoblasts and stroma after a short period of ethanol exposure. There is an increase in oxidative stress, primarily involving the nitric oxide pathway, in the trophoblasts as well as DNA damage in the stroma. Lipid peroxidation is not acutely changed in our 2-hour exposure window.

Placental Villous Glucose Metabolism and Hormone Release Respond to Varying Oxygen Tensions

Objective: The effects of varying oxygen tensions on tissue metabolic behavior are not well understood, yet many intracellular pathways are influenced by them. In the placenta, optimal in vivo oxygen tension at the villous level is unknown. The purpose of this study was to determine effects of varying oxygen tensions on glucose metabolism and hormone from perifused placental villous explants. Methods: Placentas from term normal pregnancies (n = 8) were individually minced into villous fragments, placed into three parallel chambers for each placenta, and continuously perifused for 6 hous with nonrecirculating medium aerated with either 0%, 20% or 95% oxygen yielding mean oxygen tensions of 76 mmHg, 167 mmHg, and 543 mmHg respectively, Outflow medium was removed at regular intervals and compared to the inflow medium to determine oxygen and glucose consumption as well as lactate, lactate dehydrogenase, hCG, estradiol, and progesterone release. Results: Oxygen consumption was directly proportional to oxygen tension. Glucose consumption was lowest at low oxygen tension, while both lactate and LDH release were lowest at high oxgen tension. Both hCG and progesterone release rates lowest at high oxygen tension. Estradiol release demonstrated a trend similar to that of the other hermones although there was no statistically significant difference among the three different levels of oxygen tension. Conclusion: Varying oxygen tensions affect placental villous glucose metabolism and hormone release. Under lower oxygen tensions, glucose is metabolized through glycolysis rather than through oxidative phosphorylation and is associated with higher lactate release. Exposure to higher oxygen tensions results in reduced hCG and progesterone release. Higher oxygen tensions may be associated with tissue toxicity.

Intravillous Eicosanoid Compartmentalization and Regulation of Placental Blood Flow

Objective: To determine the roles of the eicosanoids thromboxane and prostacyclin, and their compartmentalization, in the regulation of placental blood flow. Methods: First, the sites of production of thromboxane and prostacyclin were determined within the placental villus using immunohistochemical staining for thromboxane and prostacyclin synthetase. Second, the production of both eicosanoids was studied in cultured trophoblasts and compared with that in the villous core by measuring the metabolites thromboxane B2 and 6-keto-prostaglandin F1α. Finally, eicosanoid production was assessed in intact villi after stimulation by an acute change in oxygen content, 5% to 95%. Results: Immunohistochemical staining showed that thromboxane production was primarily within the trophoblasts, whereas prostacyclin production was localized to the endothelial cells within the villi. In culture, we found preferential production of prostacyclin by the villous core cells and increased production of thromboxane by trophoblasts. Perifusion of intact villi demonstrated increased production of thromboxane by trophoblasts in response to an increase in oxygen content. Prostacyclin levels were too low to be detected. Conclusions: Placental blood flow appears to be regulated by compartmentalized eicosanoids, with thromboxane affecting primarily the maternal side of the placental circulation and prostacyclin affecting primarily the fetal side.

Alloimmune thrombocytopenia may be associated with systemic disease

Adverse outcome was encountered in a case of neonatal alloimmune thrombocytopenia after in utero platelet transfusion. This may have resulted from generalized systemic vascular endothelial damage, because several cell types, including umbilical endothelial cells, have been shown to contain surface molecules similar to the receptor containing the PlA1 antigen.

Phosphorus 31 magnetic resonance spectroscopy of perifused human placental villi under varying oxygen concentrations.

OBJECTIVE Initial phosphorus magnetic resonance spectroscopy observations on the oxygen metabolism of placental villi from normal term pregnancies are described. STUDY DESIGN Villi were suspended in medium and perifused within a custom-designed 30 mm nuclear magnetic resonance probe in a superconducting vertical nuclear magnetic resonance magnet where pH, temperature, and oxygenation were monitored. RESULTS Phosphorus resonances were observed from adenosine triphosphate, phosphomonoesters. inorganic phosphate, and phosphodiesters. No phosphocreatine signal was observed. The placental villus tissue responded to an increase in oxygen concentration of the perifusate with a rise in the adenosine triphosphate level and a concomitant decline in the inorganic phosphate and the phosphomonoester signals. CONCLUSION The changes observed reflect continuing dynamic glycolysis and oxidative phosphorylation. The absence of a phosphocreatine peak suggests that aerobic pathways not driven by creatine kinase are important for placental metabolism. Our system demonstrates dynamic oxygen metabolism in perifused viable placental villus tissue by means of magnetic resonance spectroscopy.

Comparative analysis of normal and growth-retarded placentas with phosphorus nuclear magnetic resonance spectroscopy

OBJECTIVE Phosphorus 31 magnetic resonance spectroscopy studies were carried out on placentas from normal vaginal and elective cesarean deliveries without antenatal complications and from pregnancies complicated by intrauterine growth retardation of unknown cause to determine differences. STUDY DESIGN Perchloric acid extraction was performed on frozen tissue, and quantitative analysis was carried out for well-resolved resonances representing adenosine triphosphate, sugar phosphate, inorganic phosphorus, diphosphoglycerate, glycerophosphorylethanolamine, and glycerophosphorylcholine. RESULTS Adenosine triphosphate levels were highest in the growth-retarded group. There were significantly higher levels of sugar phosphate, diphosphoglycerate, and glycerophosphorylcholine in the placentas of the growth-retarded pregnancies compared with those from normal placentas. CONCLUSION These differences may represent a response to hypoxia and an increase in the amount of blood in the placenta. The results demonstrate the utility of nuclear magnetic resonance spectroscopy for studying the pathology of abnormal placentas to gain a better understanding of the pathology and represent early steps toward in vivo spectroscopic studies of the placenta.

Phosphorus 31 magnetic resonance spectroscopy of human placenta and quantitation with perchloric acid extracts

Phosphorus 31 magnetic resonance spectroscopic studies of fresh placental tissue are reported that indicate resonances for adenosine triphosphate, inorganic phosphate, sugar phosphates-phosphomonoesters, and phosphodiesters. Perchloric acid extract methods were used to further characterize and quantitate phosphorous metabolites in term human placentas by phosphorus 31 magnetic resonance spectroscopy. The perchloric acid extracts give enhanced resolution of phosphorus signals and allow identification of other phosphorus metabolites including small amounts of phosphocreatine. Emphasis was placed on quantitating adenosine triphosphate levels in the acid extracts with the use of the external reference standard hexachlorocyclotriphosphazene in a coaxial capillary system. Adenosine triphosphate levels measured in this way ranged from 0.404 to 0.709 mumol per gram wet weight. Comparison with an internal standard method with phosphocreatine is also reported. Contribution to the measured high-energy phosphate pool from blood in the highly vascularized tissue was found to be relatively large and could range from 30% to 50% of the total adenosine triphosphate measured.