Participants

This study was approved by the institutional review board at the University of Texas Health Science Center at San Antonio (approval no. HSC20070723H). Women with uncomplicated singleton term (37-40 weeks’ gestation) pregnancies were recruited with written consent before undergoing scheduled cesarean deliveries, which routinely were scheduled at 39 weeks’ gestation. These women had had ≥1 previous cesarean delivery. They were either not a candidate for a trial of labor after a previous cesarean delivery or declined a trial of labor. Gestational age was estimated from the date of the last menstrual period and confirmed by ultrasound dating.

Prepregnancy weight was determined by the nonpregnant weight that had been stated in the participant’s medical record. If no such data were available in the medical record, we obtained the nonpregnant weight verbally from the participant, which is not a standardized method. There were 2 participants in the normal BMI group and 4 participants in the obese BMI group who did not have a documented nonpregnant weight stated in the medical record. Participants were grouped on the basis of prepregnancy BMI into a normal (BMI, 18.5-24.9 kg/m ² ) and an obese group (BMI, ≥30 kg/m ² ).

Women who smoked cigarettes, used any street drugs, or consumed alcohol were excluded from the study. Additionally, gestational diabetes mellitus and any chronic medical illnesses were exclusion criteria. All of the women were of Hispanic ethnicity. According to their medical records, all study participants were taking daily prenatal vitamins that contained 1 mg of folic acid at the time of delivery.

Sample collection

On the day of the scheduled cesarean delivery once the participant was consented, maternal blood samples were collected. After delivery, the cord was double clamped; the placenta was obtained, and blood was collected from the umbilical vein. Because an umbilical blood sample could not be obtained in some study participants, the number of umbilical vein blood samples (normal BMI, 9 women; obese BMI, 8 women) were lower than the number of maternal samples (normal BMI, 15 women; obese BMI, 13 women). Serum was prepared and frozen at −80°C until analysis of folate concentrations. The concentrations were measured on an Immulite 1000 (Siemens Healthcare Diagnostics, Deerfield, IL) according to the manufacturer’s instructions. Intra- and interassay coefficients of variation were 6.7% and 7.9%, respectively, at 700 pg/mL. Villous tissue was dissected free of decidua and fetal membranes. Samples of villous tissue were taken by random sampling and were fixed either for immunostaining or used for isolation of syncytiotrophoblast microvillus plasma membrane (MVM).

Immunohistochemistry for folate transporters

Villous tissue samples were placed in formalin or zinc fixative for 18-24 hours, dehydrated, embedded in paraffin, cut into 4-μm thick sections, and subsequently mounted on slides. Sections were incubated overnight at +4°C with polyclonal antibodies toward FR-α (Santa Cruz Biotechnology, Inc, Santa Cruz, CA), PCFT (Abcam Inc, Cambridge, MA), or RFC (Alpha Diagnostic International, San Antonio, TX), at 1:250, 1:500, and 1:50 dilution, respectively. For negative controls, primary antibodies were incubated in excess antigen peptide. Incubation with appropriate biotinylated secondary antibody was for 1 hour. Antibody binding was detected by the application of avidin-peroxidase for 1 hour, followed by incubation with chromogen diaminobenzidine plus nickel to produce a brown-black precipitate.

Preparation of placental homogenates and MVMs

Syncytiotrophoblast MVM were prepared as described previously with homogenization, Mg++ precipitation, and differential centrifugation. The final pellet was resuspended in an appropriate volume of buffer to give a final protein concentration of 5-10 mg/mL. Vesicles were aliquoted, snap frozen in liquid nitrogen, and stored at −80°C until use. MVM purity was determined as the enrichment of alkaline phosphatase activity compared with homogenates and was assessed with standard activity assays for alkaline phosphatase. Enrichment of alkaline phosphatase activity in MVM was 13.6 ± 2.4–fold in placentas from normal weight women (n = 7), which was not significantly different from a 16.8 ± 3.2–fold enrichment in placentas that were obtained from obese women (n = 7; P = .4). Only MVM preparations with an alkaline phosphatase enrichment of >10-fold were included in the study.

Western blot analysis of folate transporters

Homogenates and MVMs were diluted 1:1 with sample buffer to a final loading concentration of 35 μg with a volume of 20 μL and heated to 95°C for 5 minutes; proteins were separated with the use of Biorad 12.5% Tris-HCL, an 18-well precast gel. Proteins were transferred electrophoretically onto nitrocellulose membranes at 200 volts for 1.5 hours. The membranes were blocked in Tris-glycine–buffered saline solution that contained 0.05% Tween and 5% dry milk overnight at 4°C. After being washed in Tris-glycine–buffered saline solution, membranes were incubated in antibodies that targeted FR-α (Enzo Life Sciences, Plymouth Meeting, PA) at 1:1000 dilution, PCFT (Abcam Inc) at 1:250 dilution, or RFC (Alpha Diagnostic International) at 1:150 dilution overnight at 4°C. Membranes were incubated in secondary antibodies for 1 hour at room temperature at a dilution of 1:8000 (anti-mouse) for FR-α or at 1:10,000 (anti-rabbit) for PCFT and RFC. Bands were visualized by enhanced chemiluminescence according to the instructions of the manufacturer (Amersham Biosciences, Piscataway, NJ). The images were captured with a G:Box with GeneSnap software (Syngene, Frederick, MD).

MTHF uptake by MVMs

The methods to determine MTHF uptake in the MVMs were based on published techniques for amino acid uptake in syncytiotrophoblast plasma membrane vesicles and modified according to methods reported by Yasuda et al. In brief, MVM vesicles were preloaded by incubation in 300 mmol/L mannitol, 1 mmol/L adenosine diphosphate (ADP), and 10 mmol/L Hepes-Tris, pH 7.4, overnight at +4°C. Subsequently, MVM vesicles were pelleted and resuspended in a small volume of the same buffer (final protein concentration, approximately 5-10 mg/mL). Membrane vesicles were kept on ice until immediately before transport measurements when samples were warmed to 37°C. At time zero, 30 μL of vesicles were rapidly mixed (1:3) with the appropriate incubation buffer that contained ³ H-MTHF (Moravek Biochemicals, Brea, CA) to a final concentration of 50 nmol/L. Binding of MTHF to FR-α and transport that were mediated by RFC were determined with the use of incubation buffers that were adjusted to pH 7.4. In contrast, PCFT activity was assessed with incubation buffers that were adjusted to pH 6.0, which provided the necessary inwardly directed proton gradient. In initial time course studies, uptake of radio-labeled substrate was terminated by the addition of 2 mL of ice-cold phosphate-buffered saline solution after 5-30 seconds. For subsequent uptake studies in MVM that had been isolated from normal and obese placentas, 5 seconds (for pH 7.4) and 20 seconds (for pH 6.0) were used. After stopping the reaction, vesicles were separated rapidly from the substrate medium by filtration on mixed ester filters (0.45 μm pore size; Millipore Corporation, Bedford, MA) and washed with 3 × 2 mL of phosphate-buffered saline solution. In all uptake experiments, each condition was studied in triplicate for each membrane vesicle preparation. Filters were dissolved in 2 mL liquid scintillation fluid and counted; uptakes were expressed as picomoles per milligram of protein. Nonmediated uptake/nonspecific binding was determined in the presence of 1.5 mmol/L unlabeled MTHF. In our uptake experiments, all vesicles had 1 mmol/L ADP on the inside, which resulted in an outwardly directed gradient of ADP, which is the driving force for exchange with MTHF by RFC. Uptake mediated by RFC was assessed by the incubation of vesicles in 1 mmol/L ADP which was present both on the inside and the outside of the vesicle, thereby blocking the uptake mediated by RFC. Finally, the contribution of FR-α to specific binding/uptake was determined in vesicles that were incubated for 15 minutes in 0.2 units/mL of phosphatidylinositol-specific phospholipase C, which inhibits the function of glycosylphosphatidylinositol-linked cell surface proteins such as FR-α.

Data presentation and statistical analysis

Data are presented as means ± SEM. Statistical differences between groups were evaluated with the t test. Statistical significance was set at a probability value of ≤ .05.