Objective
The association between maternal chorioamnionitis and fetal oxidative stress has not been well established.
Study Design
A nested case control study was performed within a prospective cohort of term nulliparous women: 20 cases (intrapartum fever of >100.4°F) and 20 afebrile controls. Oxidative stress was assessed using ThioGlo-1 (TG-1; Calbiochem, San Diego, CA) fluorescent sulfhydryl detection. Median levels (± interquartile range) of protein-thiol sulfhydryls were compared.
Results
In early labor, maternal oxidative stress (lower protein sulfhydryls) was significantly higher in those women who subsequently had intrapartum fever develop (79.87 ± 22.88 vs 127.73 ± 43.79 counts/second per μg protein; P < .001). In contrast, cord serum sulfhydryls were not different between groups (75.77 ± 14.00 vs 75.04 ± 17.83 counts/second per μg protein; P = .99)
Conclusion
Our data suggest that the term human fetus is protected from maternal oxidative stress associated with intrapartum fever. However, maternal oxidative status in early labor is associated with subsequent intrapartum fever. Optimal fetal neuroprotection will require a more precise knowledge of pathogenic mechanisms.
Fetal exposure to chorioamnionitis (hyperthermia and inflammation) at term has long been accepted as a potent risk factor for neonatal encephalopathy and cerebral palsy. Chorioamnionitis accounts for between 11% and 22% of cases of cerebral palsy in near-term and term infants, and carries an odds ratio of 9.3 for otherwise unexplained cerebral palsy. The current diagnosis of chorioamnionitis is largely based on maternal fever; fundal tenderness cannot be used after epidural analgesia and purulent vaginal discharge is a subjective finding. Maternal oral temperature is the best indicator of intrauterine temperature but underestimates it by an average of 0.8°C. In turn, fetal core temperature is approximately 0.75°C higher than fetal skin/intrauterine temperature. Therefore, the widely used definition of intrapartum fever (38.0°C) is generally associated with fetal brain temperatures of 39.5°C or higher.
Term infants have a low risk of neonatal encephalopathy of approximately 0.12%. The observed risk of encephalopathy with fetal exposure to maternal fever alone is 1.13% and the risk with fetal exposure to acidosis (cord pH <7.05) alone is 1.58%. However, the combination of maternal fever and neonatal acidosis results in a substantial increase in the risk of neonatal encephalopathy to 12.5%, independent of neonatal sepsis. This observation has led to the hypothesis that maternal fever is associated with increased levels of fetal oxidative stress and depleted cellular reserves, increasing fetal susceptibility to hypoxic injury. Further, antioxidants have been shown to decrease fetal injury and provide neuroprotection in lipopolysaccharide (LPS)-based animal models of chorioamnionitis, especially when used prophylactically. These promising data have led to several ongoing Phase I/II studies of the safety and efficacy of N-acetyl cysteine (NAC), a compound with both antioxidant and antiinflammatory properties for the prevention of neonatal death, sepsis, and neuronal injury in the setting of chorioamnionitis in human subjects ( http://www.clinicaltrials.gov ). Our objective was to determine whether intrapartum maternal fever was associated with increased levels of maternal and/or fetal oxidative stress. Further, we sought to examine the relationship between maternal and fetal oxidative stress.
Materials and Methods
A nested case control study was performed. The initial cohort consisted of nulliparous women who were candidates for a trial of labor. Women with active infections, autoimmune conditions, or those taking antiinflammatory agents were excluded. Subjects were enrolled prospectively at ≥37 0/7 weeks’ gestation. Maternal blood was collected at enrollment and in early labor after the onset of regular, painful contractions. Maternal oral temperatures were collected hourly in labor. Cord blood was collected at delivery. All blood samples were processed to serum and stored at −80°C. Maternal data on gestational age at enrollment, height, weight, race, and ethnicity were collected prospectively. Intrapartum and neonatal data were collected from detailed chart review and from our research quality perinatal database (PINS). A total of 607 subjects were enrolled. Subjects with available aliquots of paired maternal and cord serum were identified. Subjects were excluded if significant hemolysis was identified visually in any of the collected samples. A nested case control study was performed by selecting 20 cases (intrapartum fever of >100.4°F) and 20 afebrile controls. Selection was random and was performed by the study coordinator who had no knowledge of the hypothesis. Sample dates ranged from Feb. 5, 2006, to March 12, 2009.
Oxidative stress was assessed through detection of protein-thiol redox status using the maleimide reagent ThioGlo-1 (TG-1; Calbiochem, San Diego, CA) that produces a highly fluorescent adduct after its reaction with sulfhydryl groups. Development of fetal oxidative stress results in oxidation of DNA, lipids, and proteins. Reduced protein thiol (sulfhydryl) relatively easily undergoes oxidation to sulfenic, sulfinic, and sulfonic state (the 2 later states are irreversible). Thus, a decrease of protein sulfhydryls is considered as an appropriate measure of oxidant stress induction. Briefly, samples for analysis were prepared using size-exclusion chromatography with BioSpin-6 microcolumn (BioRad, Berkeley, CA) to eliminate low molecular weight (<6 kDa) thiols. Then 20 μL eluent was added to 2 mL of 40 mM PB (pH = 7.4) in a quartz cuvette of spectrofluorometer (PTI, Piscatway, NJ) under constant stirring at 37°C. The emission (513 nm, excitation at 379 nm) of each sample was recorded for 1 minute (background) before and 2 minutes after an addition of 5 μM (final concentration) of ThioGlo-1. Each sample fluorescence saturation value corresponds to a concentration of free sulfhydryls. At the end of each experiment, 1 μM reduced glutathione was added to the sample to ensure that saturation was not associated with the concentration of TG-1. TG-1 fluorescence was normalized for sample protein concentration.
Statistical analysis was performed using SPSS 17.0 (SPSS Inc, Chicago, IL). Median levels (± interquartile range [IQR]) of protein-thiol sulfhydryls were compared using the Mann-Whitney U test. Paired nonparametric analysis was performed using the Wilcoxon signed ranks test. Correlation was assessed using the Spearman R test. Maternal and neonatal characteristics were compared by using the Student t test or Mann-Whitney U test as appropriate for continuous variables and Fisher’s exact test for categorical variables. A P value of < .05 was considered statistically significant. Power analysis determined that 20 samples per group would provide an 80% power to detect a 26% difference in levels of fetal oxidative stress.
Results
Maternal and neonatal characteristics for cases and controls are shown in the Table . Data on neonatal sepsis evaluation and blood culture results were missing for one neonate born to a febrile mother. No significant underlying demographic differences were noted between women who remained afebrile and those who subsequently developed intrapartum fever. Overall rates of epidural analgesia were very high in both groups. There were no differences in maternal temperature at admission or maternal temperature in early labor at the time of blood sampling. Not surprisingly, intrapartum fever was associated with labors that were a median of 3.6 hours longer. There was a significantly higher rate of meconium passage in fetuses of febrile parturients, which suggests an increase in fetal stress associated with maternal fever. There were no significant demographic differences in neonates by fever status. As expected, intrapartum fever was associated with an increased rate of neonatal sepsis evaluation and neonatal length of stay; however, no neonate had culture proven sepsis. There was 1 infant with neonatal seizures in the fever group. Although intrapartum fever is a known risk factor for neonatal seizures, the rate of neonatal seizures in this study was not statistically significant. The level of oxidative stress in this infant was less than the median oxidative stress for the remainder of the group.
| Variable | Fever group (n = 20) | Afebrile (n = 20) | P value |
|---|---|---|---|
| Maternal age, y | 25.8 ± 1.1 | 24.4 ± 1.1 | .36 |
| Race, % | .9 | ||
| White | 25 | 30 | |
| Black | 25 | 20 | |
| Hispanic | 50 | 50 | |
| Gestation at enrollment, wk | 37.9 ± 0.2 | 38.0 ± 0.17 | .61 |
| Gestation at delivery, wk | 40.2 ± 0.2 | 39.2 ± 0.2 | .27 |
| Admit dilation, cm | 3.0 (0–8.0) | 4.0 (1.0–5.0) | .34 |
| PROM before labor, % | 5 | 0 | .6 |
| GBS positive, % | 35 | 30 | .5 |
| Epidural analgesia, % | 100 | 95 | .5 |
| Maternal BMI | 30.5 ± 2.7 | 31.2 ± 1.9 | .82 |
| Admission temperature, °F | 98.3 ± 0.2 | 98.2 ± 0.2 | .93 |
| Temperature early labor, °F | 98.4 ± 0.2 | 98.0 ± 0.2 | .13 |
| Meconium, % | 55 | 15 | .02 |
| Duration of labor, min | 826 (369–1285) | 610 (308–1164) | < .001 |
| Birthweight, g | 3476 ± 125 | 3356 ± 111 | .46 |
| Male infant, % | 60 | 45 | .26 |
| 5-min Apgar <7, % | 5.3 | 5 | .74 |
| Neonatal sepsis evaluation, % | 84.2 | 15 | < .0001 |
| Positive blood culture, % | 0 | 0 | > .99 |
| Neonatal seizures, % | 5 | 0 | .52 |
| Neonatal LOS, d | 3.0 (2.0–5.0) | 2.0 (1.0–5.0) | .002 |
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