Abstract
Objective. The objective of this study was to detect fetal exposure to nonsteroidal antiinflammatory drugs (NSAIDs) by meconium analysis and to determine the relationship between fetal exposure to NSAIDs and the development of persistent pulmonary hypertension of the newborn (PPHN).
Methods. In a case-control study of the inborn and outborn nurseries of a large urban medical center, meconium was collected from 101 newborn infants (40 with the diagnosis of PPHN based on clinical or echocardiographic criteria and 61 randomly selected, healthy, term infants [control]) and analyzed for NSAIDs (ibuprofen, naproxen, indomethacin, and aspirin) by gas chromatography/mass spectrometry. The risk of developing PPHN was determined in infants who were exposed antenatally to NSAID.
Results. Infants with PPHN (n = 40) had a mean gestation of 38.9 weeks and birth weight of 3524 g, which were similar to the those of the control group (n = 61). However, the incidence of low Apgar scores (≤6) at 1 minute and 5 minutes was significantly higher in the PPHN group than in the control group. The diagnoses associated with PPHN were primary PPHN (25%), meconium aspiration syndrome (35%), respiratory distress syndrome (20%), low Apgar score/asphyxia (12.5%), and pneumonia/sepsis (8%). Mean duration of ventilator support for the PPHN group was 11 days. Nitric oxide (NO) was given to 19 infants (47.5%) for a mean duration of 25.4 hours. Fourteen of the 19 infants who were treated with NO (74%) required extracorporeal membrane oxygenation, and 2 died. The overall incidence of positive NSAID in meconium in the study population (n = 101) was 49.5%: 22.8% were positive for ibuprofen, 18.8% for naproxen, 7.9% for indomethacin, and 43.6% for aspirin. There was poor agreement (Cohen's κ = 0.09) between maternal history of NSAID use and NSAID detection in meconium. PPHN was significantly associated with 1) the presence of at least 1 NSAID in meconium (odds ratio [OR] = 21.47; 95% confidence interval [CI] = 7.12–64.71) or 2) the presence in meconium of aspirin (OR = 8.09; 95% CI = 3.27–20.10), ibuprofen (OR = 12.89; 95% CI 3.93–42.32), or naproxen (OR = 3.31; 95% CI = 1.17–9.33). By logistic regression analysis, low Apgar scores at 1 and 5 minutes and the antenatal exposure to aspirin, naproxen, and ibuprofen were significantly associated with PPHN and treatment with inhaled NO or extracorporeal membrane oxygenation.
Conclusion. We confirm by meconium analysis the results of previous studies that demonstrated that the use of NSAIDs during pregnancy, particularly aspirin, ibuprofen, and naproxen, is high; is grossly underestimated by maternal history; and is significantly associated with PPHN. Thus, the easy access to over-the-counter NSAIDs of pregnant women should be reevaluated, and the potential dangers of these drugs to the newborn infant should be more effectively promoted.
Persistent pulmonary hypertension of the newborn (PPHN) has been associated with antenatal exposure to nonsteroidal antiinflammatory drugs (NSAIDs).1 NSAIDs (ibuprofen, indomethacin, naproxen, and aspirin) are cyclooxygenase inhibitors that can reduce or inhibit arachidonic acid release and block the synthesis of prostaglandins and thromboxane, which are involved in maintaining ductal patency and regulation of pulmonary vasculature.2In fetal lambs, mechanical occlusion of the ductus arteriosus before birth reproduces the hemodynamic and structural features of PPHN.3,4 Pulmonary vascular resistance in the newborn lamb remains high after prenatal ligation of the ductus arteriosus.3,4 Similarly, surgical ligation of the ductus arteriosus has been shown to cause structural remodeling of the peripheral pulmonary vascular bed as evidenced by an increase in the proportion of partially and fully muscularized pulmonary arteries at the level of the terminal bronchiole and within the acinus.4 The antenatal exposure to NSAIDs also has been shown in animal studies to produce similar changes of ductal constriction, increase in pulmonary arterial smooth muscle thickness, and pulmonary arterial hypertension.5–11
The occurrence of PPHN in infants has been linked to antenatal NSAID exposure in the mother. Perkin et al12 observed significantly higher salicylate levels in the serum of infants with PPHN without right-to-left ductus shunt, indicating that the ductus arteriosus may have closed antenatally. Postmortem studies of 2 infants whose mothers received indomethacin or salicylates during pregnancy showed pulmonary arteriolar muscularization.10 PPHN also has been reported to be associated with prenatal exposure to naproxen and indomethacin.13–16
The true incidence of maternal use of NSAIDs, however, is not known. Most studies have relied on maternal history. In a structured interview study, Van Marter et al17 showed a significant association between PPHN and maternal intake of NSAIDs, although only 6% of the mothers admitted to NSAID intake. In the study by Perkin et al,12 history of salicylate ingestion was positive only in 2 of 6 (33%) mothers whose infants had PPHN and high salicylate serum concentrations. The inaccuracy of maternal history may be attributable to a recall problem or the failure of the mother to recognize the presence of NSAIDs in multiingredient over-the-counter medications.18
Meconium drug analysis is a new method for identifying in utero exposure of infants to a number of illicit and legal drugs.19–21 Compared with maternal history, meconium drug analysis has been shown to be more sensitive and more specific.21 We, therefore, designed this study with the following objectives: 1) to develop a method for detecting NSAID in meconium using gas chromatography/mass spectrometry (GC/MS) and 2) to determine the risk of antenatal exposure to NSAIDs among infants who develop PPHN after birth.
METHODS
GC/MS Assay
The GC/MS method to analyze the NSAIDs (aspirin, ibuprofen, indomethacin, and naproxen) in meconium is briefly described as follows. For each infant, several meconium samples were collected either on the first day or as soon as the infant passed meconium. The meconium samples from each infant were pooled and stored at −15°C until the time of analysis. For analysis, the meconium was thawed and mixed well and a 0.5-g aliquot was obtained and suspended in 5 mL of buffered 25% methanol (pH = 7). The mixture was vortexed and centrifuged. The supernatant was recovered and centrifuged in a millipore system. The ultrafiltrate was transferred to an extraction column (ZSDAU020, United Chem Tech, Bristol, PA), washed with 3 mL of water and 1 mL of 1 m of acetic acid, and then allowed to dry. The drugs were eluted using hexane:ethyl acetate (50:50). The eluate was evaporated to dryness with nitrogen and reconstituted with 50 μL of ethyl acetate. The sample was transferred to an autosampler vial and derivatized with 50 μL of n-methyl,n-trimethyl, silyltrifluoroacetamide at 60°C. Two μL of the sample were injected into a Hewlett Packard 5890 gas chromatograph with a 5971A mass spectrometer (Hewlett Packard, Atlanta, GA). With the use of spiked meconium samples, the recovery of drugs was 96% for aspirin, 92% for ibuprofen, 100.8% for indomethacin, and 124% for naproxen. The precision of the assay for the 4 drugs showed an interassay and intraassay coefficient of variability that ranged between 2.2% and 8.8%.
Participants
From January 1996 to September 1997, meconium was collected from infants who had received the diagnosis of PPHN and who were admitted to the neonatal intensive care units of Hutzel Hospital and Children's Hospital of Michigan, which serve an inborn and an outborn infant population, respectively. The study was approved by our institutional review board. The criteria for diagnosis of PPHN were gestational age ≥34 weeks with clinical and/or echocardiographic evidence of PPHN. The clinical criteria consisted of Pao2<100 in 100% oxygen with significant lability in the infant's oxygen saturation in the absence of cardiac disease and echocardiographic criteria that included predominant right-to-left or bidirectional shunting at the ductus arteriosus or foramen ovale or pulmonary artery pressure greater than two thirds of systemic pressure as estimated by Doppler insonation of the tricuspid jet. All echocardiograms were read by a pediatric cardiologist. Exclusion criteria were lethal congenital anomalies; structural congenital heart disease except presence of patent ductus arteriosus (PDA) or patent foramen ovale; structural gastrointestinal tract abnormality that could interfere with meconium passage; and congenital abnormalities such as congenital diaphragmatic hernia, Potter's syndrome, and pulmonary hypoplasia. The control group consisted of randomly selected, normal, healthy infants who had a gestation of ≥34 weeks, were born at Hutzel Hospital, and had an uncomplicated neonatal course. The majority of infants for the control and PPHN groups were obtained during the same period; however, an additional 10 infants in the control group were recruited later, after preliminary analysis of the case-control data revealed that there were significantly more infants in the PPHN group that were higher in birth weight and were meconium-stained.
Statistics
Descriptive statistics were performed on the variables, and comparisons between the PPHN group and the control were done by the Student's t test or Pearson's χ2analysis, as well as a measurement of risk by the odds ratio (OR) and its 95% confidence interval (CI). Variables that were significantly (P < .05) associated with PPHN were analyzed further using forward, stepwise, logistic regression analysis. The degree of agreement between antenatal use of NSAIDs by maternal history or meconium analysis was determined by Cohen's κ.
RESULTS
A total of 101 infants were enrolled in the study: 40 in the PPHN group and 61 in the control group. Thirty-five infants in the PPHN group had an echocardiogram: 17 (48.6%) showed a PDA with evidence of significant right-to-left or bidirectional shunt in 13 infants. Among the 5 infants with PPHN and no echocardiogram, 2 had perinatal asphyxia, 2 had primary PPHN, and 1 had meconium aspiration.
As shown in Table 1, the maternal profile was similar in the PPHN and control groups except for maternal race. This is because the control group was obtained from Hutzel Hospital, where the proportion of black, pregnant women is high (84%). The neonatal profiles of infants in the PPHN and control groups were similar except for significantly lower 1- and 5-minute Apgar scores in the PPHN group (Table 2). The diagnoses that were associated with PPHN included meconium aspiration (35%), idiopathic or primary PPHN (25%), respiratory distress syndrome (20%), low Apgar score/asphyxia (12.5%), and pneumonia or group B streptococcal sepsis (7.5%). Among the 40 infants with PPHN, 10 infants required only hood oxygen, but at 100% concentration, and the infants exhibited marked lability in oxygen saturation, particularly during the first 24 hours of life. Thirty infants required a ventilator; the mean duration of ventilator support was 11 days. Nineteen of these infants (47.5%) required inhaled nitric oxide (NO), and 14 infants (75%) subsequently were placed on an extracorporeal membrane oxygenator (ECMO) and 2 died (Table 3).
Maternal Profile
Neonatal Profile
Ventilatory Management of Infants With PPHN (N = 40)
PPHN was not significantly associated (P > .10) with meconium-stained amniotic fluid, prolonged rupture of the membranes, maternal group B streptococcal colonization, mode of delivery (vaginal vs cesarean section), infant's sex, or race. Conversely, PPHN was significantly (P < .01) associated with a low (≤6) 1- or 5-minute Apgar score and the presence of NSAIDs in meconium, particularly aspirin (OR = 8.09; 95% CI = 3.27–20.01) and ibuprofen (OR = 12.89; 95% CI = 3.93–42.32; Table 4). There was no significant association between PPHN and indomethacin use (OR = 2.76; 95% CI = 0.62–12.27). By stepwise, logistic regression analysis, PPHN was associated with low (≤6) 1-minute Apgar score and in utero exposure to aspirin or ibuprofen (Table 5). The severity of PPHN can be deduced from the extent of the treatment given, eg, use of inhaled NO or ECMO. The use of inhaled NO was significantly associated with a low (≤6) 5-minute Apgar score and fetal exposure to aspirin or naproxen. The use of ECMO was significantly associated with a low 5-minute Apgar score and fetal exposure to ibuprofen or naproxen (Table 5). Only a few infants (n = <10) had 2 or more NSAIDs in their meconium, which precluded further analysis on whether fetal exposure to >1 NSAID further increased the risk for PPHN.
Association Between PPHN and NSAIDs in Meconium
Logistic Regression Analysis Showing Factors That Were Significantly Associated With PPHN and the Use of Inhaled NO or ECMO in Newborn Infants
In 101 meconium samples, 49.5% were found to be positive for NSAIDs: 43.6% positive for aspirin (GC/MS analysis of aspirin was specific for acetylsalicylic acid; thus, the presence of other forms of salicylate could not be ascertained from this study), 7.9% positive for indomethacin, 22.8% positive for ibuprofen, and 18.8% positive for naproxen. In contrast, by maternal history, only 1% had a history of aspirin use, 1.6% of indomethacin use, 12.8% of ibuprofen use, and 11.7% of naproxen use. Thus, the degree of agreement between exposure to NSAIDs as detected by meconium analysis or maternal history was very low (Cohen's κ = 0.09).
There was a wide concentration range of the NSAIDs in the positive meconium samples (mean ± standard deviation): aspirin (315.5 ± 1448.6 ng/mL), indomethacin (4591.9 ± 5286.2 ng/mL), ibuprofen (702.9 ± 947.4 ng/mL), and naproxen (434.7 ± 905.3 ng/mL). By forward, stepwise, logistic regression analysis, there was a significant association between the concentrations of ibuprofen in meconium and PPHN (P < .003) and between the concentration of naproxen in meconium and the use of ECMO or NO (P < .003).
DISCUSSION
PPHN occurs at a rate of approximately 1:600 to 1:1500 live births. The predisposing factors to this disorder are multiple, eg, meconium aspiration, asphyxia, respiratory distress syndrome, and group B streptococcal pneumonia.22 The role of NSAIDs as a predisposing factor to the development of PPHN has been previously suggested in several studies.12,13,15–17 Our study, which used meconium analysis to demonstrate NSAID exposure in the fetus, has confirmed these observations. Eighteen infants with PPHN did not show a PDA on echocardiogram, which suggests the role of in utero ductal constriction by NSAID in the pathogenesis of PPHN.12
The use of NSAIDs during pregnancy is common because the exposure of the public to these medications through the print and television media is widespread.23,24 Although the labels on these drugs provide adequate warning of the potential harm to the fetus if these drugs are used during pregnancy, it is likely that the warnings are ignored. One study showed that the rate of exposure of mother–child pairs to different drug groups was highest with analgesics and antipyretic drugs (32% in the first trimester) and that NSAIDs were one of the most widely used agents during pregnancy.25 The NSAIDs are used for their antiinflammatory, analgesic, or antipyretic properties and are available over the counter. Indomethacin is the only exception and usually is available only by prescription. This may explain why, in this study, fetal exposure to indomethacin was very low. In many instances, NSAIDs may unknowingly be taken by mothers through multiingredient, over-the-counter medications. For example, Pepto Bismol (Procter & Gamble, Cincinnati, OH) contains bismuth subsalicylate, which, if hydrolyzed and absorbed in the gastrointestinal tract, results in significant serum salicylate levels.18 NSAIDs have been used for other purposes during pregnancy. Indomethacin is used as a tocolytic drug or to decrease fetal urine output in cases of polyhydramnios.26–28Low-dose aspirin has been used for the prevention of pregnancy-induced hypertension,29–31 and naproxen has been used as a tocolytic agent.16 However, reports of PPHN occurring under these conditions are few.28 The NSAIDs cross the placenta readily and have a prolonged half-life in the fetus.
Of the various NSAIDs identified in meconium, aspirin, ibuprofen, and naproxen were significantly associated with the development of PPHN. However, severe PPHN, which required ECMO treatment, was significantly associated with naproxen or ibuprofen. Whether these 2 drugs are more potent cyclooxygenase inhibitors that result in a more severe form of PPHN remains to be elucidated. The lack of association between indomethacin use and PPHN came as a surprise, because the adverse effects of indomethacin on the fetal pulmonary arterioles and ductus arteriosus are well-known.7–11 It is likely that our failure to observe an association between indomethacin and PPHN may be attributable to the small number of infants in the study who were exposed to indomethacin (n = 8). As mentioned, because indomethacin is not an over-the-counter medication, pregnant women's access to this drug is more limited. Despite our findings of a high frequency of fetal exposure to NSAIDs (18.8%–43.6%), we noted a low frequency of exposure to 2 or more drugs (10%). We cannot adequately explain this observation.
Our rate of pregnant women's reporting the use of NSAIDs during pregnancy was low. Although such information was not obtained through a structured interview, the low rate of NSAID use by maternal history likely is attributable to problems of drug recall or the difficulty in recognizing NSAIDs that are marketed under various trade names or as part of multiingredient pain medications.12,17 This study, therefore, reinforces the usefulness of meconium analysis as a sensitive test to identify drugs that the fetus may have been exposed to during gestation.
Drugs are deposited in meconium through bile secretion or through fetal swallowing of drugs in the amniotic fluid.32 Because meconium is formed as early as the 12th week of gestation and normally is not excreted by the fetus until after birth, meconium analysis offers a wide window for detecting fetal exposure to drugs. Many methods have been used to analyze drugs in meconium.21,33,34 The use of GC/MS in this study was designed to attain a high degree of sensitivity and specificity in our assay. Our recovery studies using spiked meconium demonstrated that our method for NSAID analysis in meconium was optimal. The slightly high recovery of naproxen (125%) is probably attributable to 1) a priori presence of a small amount of naproxen in meconium that was spiked with drugs for the recovery study and/or 2) an error in the weighing of naproxen that was spiked in meconium. In either case, this does not significantly affect the validity of our study, because GC/MS is a test with very high specificity. Unlike immunoassays, GC/MS does not use antibodies; thus, cross-reaction with other compounds is avoided.
CONCLUSION
We confirm by meconium analysis what has been suggested by previous studies: that NSAID use during pregnancy is significantly underestimated by maternal history and that the association between NSAID use during pregnancy—particularly aspirin, ibuprofen, and naproxen—and PPHN is high. Thus, the easy access of over-the-counter NSAIDs to pregnant women should be reevaluated, and the potential dangers of these drugs to the newborn infant should be more effectively promoted.
ACKNOWLEDGMENT
This work was supported in part by a FIRST Award from the National Heart, Lung, and Blood Institute (HL44533) to Dr Konduri.
Footnotes
- Received November 20, 1999.
- Accepted July 3, 2000.
Reprint requests to (E.M.O.) Department of Pediatrics, Hutzel Hospital, Wayne State University, 4707 St Antoine, Detroit, MI 48201.
- PPHN =
- persistent pulmonary hypertension of the newborn •
- NSAID =
- nonsteroidal antiinflammatory drug •
- GC/MS =
- gas chromatography/mass spectrometry •
- PDA =
- patent ductus arteriosus •
- OR =
- odds ratio •
- CI =
- 95% confidence interval •
- NO =
- nitric oxide •
- ECMO =
- extracorporeal membrane oxygenation
REFERENCES
- Copyright © 2001 American Academy of Pediatrics
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