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ORIGINAL RESEARCH |
From the Department of Obstetrics and Gynecology and The Collaborative Biostatistics Center, The Cleveland Clinic Foundation, Cleveland, Ohio.
Address reprint requests to: Janet M. Burlingame, MD, The Cleveland Clinic Foundation, Department of Obstetrics and Gynecology, Section of Maternal–Fetal Medicine, 9500 Euclid Avenue, Desk M-66, Cleveland, OH 44195; E-mail: burlinj{at}ccf.org.
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ABSTRACT |
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METHODS: Amniotic fluid was collected from a total of 26 nonsmoking patients. Nine specimens were collected in the third trimester of pregnancy, and 17 specimens were collected in the second trimester. Amniotic fluid reactive oxygen species and total antioxidant capacity levels were determined by chemiluminescence and spectrophotometric assays, respectively. Reactive oxygen species and total antioxidant capacity levels were established and then compared for advancing gestational ages and estimated fetal weights or neonatal weights.
RESULTS: Reactive oxygen species levels were present in some but not all specimens, and total antioxidant capacity was present in all specimens. Total antioxidant capacity but not reactive oxygen species levels increased from the second to the third trimester (347.0 mmol/L versus 776.0 mmol/L, P < .001). There was a positive Spearman correlation between total antioxidant capacity and gestational age (r = 0.72, 95% confidence interval 0.43, 1.0, P < 001) and between total antioxidant capacity and estimated fetal weights or neonatal birth weights (r = 0.70, 95% confidence interval 0.40, 1.0, P < .001). There was no correlation between reactive oxygen species and advancing gestational age or weight.
CONCLUSION: Total antioxidant capacity levels are present in amniotic fluid at least as early as the second trimester and increase with advancing gestational age and fetal or neonatal weights. Reactive oxygen species levels are not necessarily present in amniotic fluid, and they do not appear to be influenced by gestational age or estimated fetal or neonatal weights.
Free radicals and reactive oxygen species play a number of significant and diverse roles in reproductive biology.1 Pregnancy represents a complex state in reproductive biology in which the mother and the fetus may both contribute to the oxidative stress and production of reactive oxygen species. Oxidative stress as reflected by free radicals and reactive oxygen species has been measured in maternal and fetal serum, but has yet to be measured in amniotic fluid. Reactive oxygen species are highly reactive oxygen radicals such as superoxide anion, hydroxyl radical, hydrogen peroxide, and the hypochlorite radical. They are cytotoxic in that they cause peroxidation of membrane phospholipids, which results in increased membrane permeability, loss of membrane integrity, enzyme inactivation, and structural damage to deoxyribonucleic acid, all of which lead to cell death.2,3
Antioxidants, on the other hand, counteract the effects of these free radicals and thereby protect cell membranes from lipid peroxidation.4 The ability of a tissue or fluid to buffer the effects of reactive oxygen species is called total antioxidant capacity.5
Oxidative stress occurs when there is an excessive production of reactive oxygen species or when total antioxidant capacity decreases.6 Oxidative stress induced by reactive oxygen species has been implicated in many intrinsic and extrinsic pregnancy-associated problems. Intrinsic pregnancy-associated problems that are affected by reactive oxygen species include embryopathy from diabetes or substance abuse, preterm premature rupture of membranes secondary to bacterial infection or other insults, labor, and preeclampsia.6–11 Venous cord blood has also been shown to contain depressed levels of total antioxidant capacity in premature infants.12 Extrinsic pregnancy-associated concerns that may be affected by reactive oxygen species include smoking and lung disease.13–15
Reactive oxygen species and oxidative stress have also been associated with poor fetal growth in animal studies. For example, oxidative stress was associated with the presence of intrauterine growth retardation and fetal weights in rat and pig models.16–20
Despite these documented pathologic associations, reactive oxygen species levels and total antioxidant capacity have not been measured in amniotic fluid, and the significance of their presence or absence remains unclear. The goal of this prospective observational study was to determine whether reactive oxygen species and total antioxidant capacity are present in amniotic fluid and, if so, measure their levels at varied gestational ages and at varied estimated fetal weights (second trimester) or birth weights (third trimester).
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MATERIALS AND METHODS |
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To be included in this study, the participants had to provide at least 2 mL of amniotic fluid, and had to answer a small series of medical history questions regarding the presence or absence of hypertension and diabetes, smoking status, and use of prenatal or other vitamins. Amniotic fluid was collected from smokers and nonsmokers as part of a larger study, but in the analysis presented here, only nonsmokers were included. Patients were excluded if emergent delivery was necessary, if they refused to be enrolled in the study, or if the gestational age of their fetus could not be accurately determined. An average daily vitamin dose was calculated by averaging the dose of vitamins E or C in the brand of vitamins reported by the patient at time of enrollment.
Gestational age was based on the last menstrual period or by a second-trimester ultrasound when the last menstrual period was unknown or differed by more than 10 days from the findings of a second-trimester ultrasound. Fetal weights were estimated using Hadlock equations and measurements obtained from a level II ultrasound at the time of the genetic amniocentesis. Neonatal birth weights were recorded for the patients enrolled in the third trimester. The interval between amniocentesis and the determination of birth weights for the third trimester patients was less than 48 hours in all patients.
All amniotic fluids were collected in sterile tubes at room temperature. During cesarean delivery, a sterile needleless syringe was used to collect the fluid upon entering the amniotic cavity and after the myometrium had been bisected sharply and distinctly from the amniotic-chorionic membrane. We discarded the samples that were visibly contaminated with blood. The specimens were transferred within 1 hour of collection to a laboratory and processed within 15 minutes. The amniotic fluid was divided into two aliquots; the first aliquot was used as a neat sample, and the second aliquot was centrifuged (500g, 7 minutes) to remove the cellular contamination. Also, 200 µL from the supernatant was frozen immediately at -70C for any further total antioxidant capacity measurement.
Levels of reactive oxygen species in the neat and centrifuged amniotic fluids were measured using a Berthold luminometer (Autolumat LB 953; Wallace Inc., Gaithersburg, MD). Extreme care was taken to minimize the time between the aspiration of amniotic fluids and reactive oxygen species measurement to reduce the variability in the results. As degradation starts to occur within 2 hours, all specimens were analyzed for reactive oxygen species within 1.5 hours of collection. Aliquots of 400 µL of neat and centrifuged amniotic fluids were prepared in duplicate along with the blank and the controls. Levels of reactive oxygen species were determined by chemiluminescence assay using luminol (5-amino-2, 3, -dihydro-1, 4-phthalazinedion; Sigma Chemical Co., St. Louis, MO) as a probe. Luminol is a highly sensitive chemiluminescent probe, and it reacts with a variety of reactive oxygen species such as oxygen-centered radicals (for example, superoxide radical, hydroxyl ion, nitric oxide, peroxynitrite), reactive nonradicals (for example, hydrogen peroxide singlet oxygen, hypochlorus acid), and nonoxygen-centered radicals (for example, thiyl, trichloromethyl). The reaction of luminol with reactive oxygen species causes light emission that is proportional to the level of reactive oxygen species generation. After adding 10 µL of luminol (5 mM), the measurements were recorded for 15 minutes in integration mode, and the results were expressed as x 106 counted photons per minute. The background luminescence was determined using the blank without the luminol. All assays were performed in the dark.21 The intraassay coefficient of variation was 2% with an intraassay reliability of 93.8%. The interassay coefficient of variation was not significant, equaling 0.9% (P > .8).
Amniotic fluid was stored at -20C. Total antioxidants were measured at 37C with a kit based on the method of Miller et al (Randox Laboratories Cat. No NX 2332; Crumlin Co., Atrium, UK).12 This method arose from the finding that when the chromogen 2,2'-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) was incubated with hydrogen peroxide and a peroxidase (metmyoglobin), the relatively long-lived radical cation 2,2'-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) · + is formed, which has a relatively stable blue-green color and can be measured at 600 nm. Antioxidants in the sample suppress the color production to a degree that is proportional to their concentration. An antioxidant ranking was established based on the reactivity relative to a 1.0 mmol/L of Trolox (an 
-tocopherol analogue) standard, otherwise referred to as the Trolox equivalent antioxidant capacity of amniotic fluid. Results were expressed in mmol/L. The precision of this assay has been previously estimated using an intraassay coefficient of variation (0.54–1.59%) and interassay coefficient of variation (3.6–6.1%).12
At the time of enrollment, participants were asked to report the brand of vitamin that they were taking. The average daily intakes of supplemental vitamins E and C were calculated by averaging the dose of these vitamins found in the prenatal vitamins taken by the study participants.
The Spearman rank order correlation coefficient (with 95% confidence intervals) was used to assess the relationship between reactive oxygen species, total antioxidant capacity, gestational age, and weight.22 Correlations between reactive oxygen species or total antioxidant capacity and weight were made with and without partialling out gestational age. Differences in reactive oxygen species and total antioxidant capacity between second and third trimesters were assessed with the Wilcoxon exact rank-sum test. A significance level of 0.05 was used for each hypothesis, and the statistical tests were two sided. With the 26 subjects in this study, there was 80% power to detect correlations of 0.50 or larger at the 0.05 significance level. SAS statistical software (SAS Institute Inc., Cary, NC) was used to perform the statistical analysis.
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RESULTS |
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).
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). There was no statistically significant correlation detected between reactive oxygen species levels and gestational age or estimated fetal weight and birth weight (Table 3).
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). There was a statistically significant (P < .001) correlation between total antioxidant capacity levels and gestational age (r = 0.72) and weight (estimated fetal weights and neonatal birth weights, r = 0.70). In fact, the 95% confidence intervals of the Spearman correlation coefficients for gestational age and weight were almost identical at 0.43, 1.0 and 0.40, 1.0, respectively (Table 3 and Figure 1). The Spearman correlation coefficient for the relationship between gestational age and weight was 0.97 (P < .001) (Table 3).
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DISCUSSION |
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This study demonstrates that total antioxidant capacity, but not reactive oxygen species, was consistently present in all amniotic fluid specimens regardless of gestational age. In addition, total antioxidant capacity appeared to be positively correlated with gestational age and estimated fetal or neonatal weights. The reactive oxygen species levels showed neither any consistency nor any statistically significant association with gestational age or weights.
Now that we have documented the existence of reactive oxygen species and total antioxidant capacity in amniotic fluid and a dynamic change in the levels of total antioxidant capacity during advancing gestation, researchers can continue to broaden the study of oxidative stress in pregnancy. Understanding the origin of reactive oxygen species and total antioxidant capacity in the amniotic fluid will help define the role that oxidative stress potentially plays in preterm premature rupture of membranes, intrauterine growth retardation, preeclampsia, and labor. Whether the origin of total antioxidant capacity is the fetus, the placenta, the mother, or a combination of these sources is unclear. Localizing the site of production of reactive oxygen species and total antioxidant capacity will also help in understanding the role it plays in both normal homeostasis and disease states.
There are multiple origins of amniotic fluid and its various components. Considering that most of the amniotic fluid in the second and third trimesters comes first from fetal urine and secondly from fetal lung secretions, the fetus should be considered as a potential source of reactive oxygen species and total antioxidant capacity. Our data also support this supposition because the total antioxidant capacity levels positively correlate with both gestational age and with fetal weight. This supports the theory that the fetal age and/or fetal size may be at least partially responsible for the level of total antioxidant capacity. However, amniotic fluid is also created by active and passive intramembranous exchange within the chorionic plate and the amniotic-chorionic membranes and transmembranous exchange across the uterine wall and amniotic-chorionic membranes.24 Additional research will help identify the actual source of total antioxidant capacity and reactive oxygen species production in amniotic fluid. Future research may also help distinguish whether total antioxidant capacity is determined by the chemical maturity of the fetus with advancing gestational age or if it is simply a matter of larger fetal mass with increasing estimated fetal or neonatal birth weights. It would also be interesting to break down total antioxidant capacity into components and separately measure antioxidants such as transferrin and uric acid, which would probably account for different degrees of importance to the overall level of amniotic fluid antioxidant capacity. With these individual measurements, one might identify separate origins for different antioxidants.
Potential confounders in this study include anything that can alter the oxidative environment of the amniotic fluid such as smoking. Therefore, smokers were eliminated from this analysis. Because smoking can cause oxidative damage in the nonpregnant state, it is possible to theorize that oxidative stress may be one of the contributors to cigarette or other smoking toxicity to the fetus. Tobacco smoke has already been associated with increases in oxidative stress in nonpregnancy-related tissues. For example, tobacco smoke exposure has been shown to increase oxidative stress in rat testes and in human neutrophils.13,14 Tobacco smoke has also been implicated as a cause of oxidative stress in human lung diseases such as emphysema, cancer, and chronic obstructive pulmonary disease.14,15 Like smoking, diseases related or unrelated to pregnancy may alter the amniotic fluid oxidative environment. Interestingly, it is these potential confounders that may have a clinical interest in elucidating the pathophysiology of associated pregnancy complications.
Vitamin intake could also be a potential confounder by artificially increasing antioxidant levels. Vitamins E and C are extrinsic antioxidants and are ingested by the majority of pregnant women. Vitamin supplementation was reported in 69% of all our recruited patients. Therefore, in this study, we did not exclude the patients who used vitamins, and this could prove to be a limitation of the study.
Finally, the study was limited by the sample size. With only 26 patients, we cannot definitively state that there is no difference between reactive oxygen species levels in different trimesters or that there is no correlation between reactive oxygen species and gestational age and fetal weight.
Our analysis of amniotic fluid with respect to oxidative stress and buffering capacity raises many interesting points and questions. For example, is it coincidental that the frequency of conditions that have been previously associated with oxidative stress, such as labor, fetal growth restriction, and preeclampsia, increases near term? This would teleologically help explain our observation of increased total antioxidant capacity levels near term. Our attempt to establish the presence of total antioxidant capacity in the amniotic fluid and its increase with increasing fetal weight and gestational age lays the groundwork for future research into the potential contribution of oxidative stress in the pathophysiology of multiple pregnant disease states and even labor initiation. Amniotic fluid will provide an easily accessible medium in which to study oxidative stress during an ongoing gestation. By increasing the patient sample size, it will be possible to determine any impact that smoking or the disease states, such as hypertensive disorders, diabetes, growth restriction, and premature rupture of membranes, may have on oxidative stress and buffering capacity as measured in the amniotic fluid or vice versa. Perhaps future research regarding oxidative stress reflected in the amniotic fluid may even help predict the onset of term and preterm labor.
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Footnotes |
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The authors thank Elliot Philipson, MD, Stephen Emery, MD, and Brian Clark, MD.
doi:10.1016/S0029-7844(02)03069-7
Received March 21, 2002. Received in revised form July 31, 2002. Accepted August 15, 2002.
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