Objective
To compare endothelial nitric oxide synthase expression and capillary density (CDS) in placentas exposed to single or multiple courses of betamethasone.
Study Design
Placental specimens exposed to single vs repeat courses of betamethasone were analyzed through immunohistochemistry and digital image quantification for endothelial nitric oxide synthase and CD34. Quantified endothelial nitric oxide synthase staining, calculated capillary density, ratio of endothelial nitric oxide synthase to capillary density, and clinical characteristics were compared. Linear regression was performed with these as dependent variables.
Results
Mean and maximum capillary density were increased ( P = .013 and .005) and the ratio of endothelial nitric oxide synthase to capillary density decreased ( P = .016) in specimens exposed to 4 courses of betamethasone compared with 1 to 3 courses. Exposure to 4 courses of betamethasone was associated with increased capillary density, but not with endothelial nitric oxide synthase expression.
Conclusion
Exposure to 4 courses of betamethasone is associated with increased placental capillary density. The placental effects of multiple courses of betamethasone are unrelated to endothelial nitric oxide synthase expression.
Multiple courses of antenatal betamethasone have been shown to result in decreased birthweight and neonatal head circumference. However, the mechanism of this effect remains unclear.
In studies examining fetal growth restriction, it has been found that the large increase in fetal growth during the last half of pregnancy depends primarily on a corresponding increase in transplacental exchange. This occurs through coordination of angiogenesis, adaptations of vascular tone, and efficiency of exchange and is supported by significant growth of the placental vascular beds with resultant increases in uterine and umbilical blood flow. It has further been shown that the vascular density of fetal placental components increases dramatically during the last third of gestation as compared with maternal components, which have been shown to have a consistent slow increase throughout gestation.
Endothelial nitric oxide synthase (eNOS) is an important component of regulation of vascular tone and blood flow and has been linked to fetoplacental growth and development. Several authors have postulated that this is a result of eNOS-mediated nitric oxide release from the fetal tissues and is supported by immunoreactive and in situ hybridization demonstration of eNOS expression by extravillous trophoblast cells.
In addition, antenatal glucocorticoid exposure in primates has been shown to significantly reduce eNOS protein as well as other indices of eNOS function. This effect has been seen in both the syncytiotrophoblast as well as the vascular endothelium.
The objective of this study was to compare eNOS expression and capillary density in placentas from pregnancies exposed to multiple or single courses of betamethasone. It was our hypothesis that betamethasone exposure influences fetal growth through a nitric oxide-mediated mechanism.
Materials and Methods
Study design
This study is an analysis of placental specimens collected during the conduct of a randomized multicenter trial that compared the effect of administering single vs weekly repeat courses of antenatal betamethasone in preventing neonatal morbidity. The trial was conducted between March 2000 and April 2003 at participating centers of the Eunice Kennedy Shriver National Institute of Child Health and Human Development ( Appendix ), Maternal-Fetal Medicine Units (MFMU) Network. The primary study has been previously described. Participants in the trial were pregnant women between 23 weeks 0 days and 31 weeks 6 days, who were at risk for spontaneous preterm delivery. Women with preterm premature rupture of the membranes (PPROM) before randomization, confirmed fetal lung maturity, chorioamnionitis, a major fetal anomaly, nonreassuring fetal status, systemic corticosteroid use during the current pregnancy, or insulin-dependent diabetes were excluded.
Patients were randomly assigned to receive weekly courses of betamethasone or placebo, 1 week after receiving a single full course of corticosteroids (betamethasone or dexamethasone). Each course consisted of 2 injections of betamethasone 12 mg (as 6 mg betamethasone sodium phosphate and 6 mg betamethasone acetate) repeated once in 24 hours or matching placebo. Initially, patients received courses until delivery or 33 weeks 6 days’ gestation, whichever was sooner. After 67 patients had been enrolled, the number of courses (not including the qualifying course) was limited to 4 because of difficulty in recruitment and because of accumulating literature suggesting possible harmful effects of multiple courses.
Placentas were collected at the time of delivery. For this analysis, placental specimens from patients with singleton pregnancies were evaluated.
Placental specimen preparation
Specific samples were obtained from each placental specimen and were formalin fixed and paraffin embedded. All samples included in this analysis were from a standardized lateral location within the placenta containing both maternal and fetal surfaces. Institutional review board approval was obtained before conducting this investigation.
After obtaining the paraffin blocks of these placental tissues, sections were cut at 4 μ, adhered to ProbeOn Plus (Nextag, San Mateo, CA) slides from a protein-free water bath and air-dried overnight. Slides were then deparaffinized and hydrated to deionized water. After HIER (Heat Induced Epitope Retrieval) in Citrate buffer pH 6.0 (eNOS/NOS type III; BD Transduction Laboratories, San Diego, CA) for 30 minutes using a rice steamer and enzymatic retrieval using Trypsin at 1 mg/mL, 4 mM CaCl 2 in 20 mM TRIS pH 7.7 for 30 minutes at 37°C, slides were washed for 5 minutes under running deionized water, then transferred to a hand-held MicroProbe slide holder (Cole-Parmer, Vernon Hills, IL). A 1X Automation Buffer (Biomeda, Foster City, CA) containing 0.1% Tween 20 to eliminate surface tension and 0.5% Casein (Sigma-Aldrich, St. Louis, MO) to blocking nonspecific background, was used both as wash buffer and antibody diluent. Serial sections were incubated with mouse monoclonal eNOS/NOS Type III (BioGenex, Fremont, CA) at 1:50 and monoclonal CD34 (Novocastra, Newcastle upon Tyne, UK) at 1:10 overnight at 4°C. After several washes with 1X buffer, slides were incubated with biotinylated anti-mouse antibodies (BioGenex) at 1:20 for 20 minutes at 37°C. After additional washes using 1X buffer, sections were incubated with a streptavidin-alkaline phosphatase complex (BioGenex) at 1:20 for 20 minutes at 37°C. The red end product was visualized using a Vector Red chromogen (Vector Laboratories, Burlingame, CA). Slides were counterstained for 5 minutes in Mayer’s hematoxylin, washed under running deionized water, dehydrated using graded alcohols, cleared through p-xylene, and mounted with Permount ( Figures 1 and 2 ). This technique is applied routinely to paraffin-embedded specimens in both clinical and research immunohistochemistry laboratories and the antibodies used in this investigation were specifically noted for use on paraffin sections. The HIER process described previously enhances this technique and consists of enzymatic retrieval of protein cross-linkages that might have occurred through formalin fixation in these tissues, allowing return to a native configuration of any proteins that may have been altered during fixation.
