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Objective To determine the effect of labour on free oxygen radical activity in the fetus, as reflected by lipid peroxide levels in umbilical cord arterial blood.
Design Prospective, observational study.
Setting Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong.
Methods Umbilical cord arterial and venous blood samples were collected from singleton term infants delivered by elective caesarean section. Base excess, PO2, pCO2, and pH were measured in both samples and compared to identify double venous samples. Cord arterial acid-base balance and concentrations of organic hydroperoxides and malondialdehyde were compared with those obtained from normal vaginal deliveries.
Results Cord arterial blood samples, obtained from cases of uncomplicated labour followed by spontaneous vaginal delivery, had significantly higher lipid peroxide concentrations than those delivered following elective caesarean section. This was most marked for malondialdehyde with a median value increased by 105%, whilst organic hydroperoxide was increased by only 27%. Of the acid-base parameters, base excess was increased by 78%, with only minimal changes in pH, PCO2, and PO2. These differences remained highly significant after including other pregnancy characteristics in multivariate analysis.
Conclusion The findings indicate that high levels of free oxygen radical activity in the fetus are a function of the labour process, as are changes in acid-base balance.
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This study included 91 women with normal, singleton pregnancies between 37 and 42 weeks gestation, delivered by elective caesarean section, in whom validated cord arterial blood samples were available. These were compared with 182 women undergoing normal vaginal delivery in the same unit.
Immediately after delivery a segment of umbilical cord was double-clamped and blood was drawn from both the umbilical artery and the umbilical vein into separate 5-mL pre-heparinised plastic syringes. All blood sampling and subsequent biochemical analyses were performed by two of the authors (W.C.C. and L.K.P.). The whole blood samples were analysed within five minutes of collection on a Ciba Corning USA) for pH, carbon dioxide (pCO2), oxygen (PO2), oxygen saturation and base excess. Acid-base parameters were compared between the paired samples using a computer program developed by Mongelli et al.8 for identifying double venous samples: samples with a negative arteriovenous pH difference < 0.02; a negative arterio-venous PO2 difference < 0.4; or a positive pCO2 difference and > 0.4 were excluded from analysis.
The plasma in the arterial blood samples was collected immediately after refrigerated centrifugation at 1000 g for 10 minutes, and then stored at -20°C prior to assay for lipid peroxide concentrations. The plasma MDA levels were estimated as reactive substances by a thiobarbituric acid addition method described by Richard et al.9 The method used for determination of organic hydroperoxides was the enzymatic technique, as described by Heath and Tappel.10
Statistical analyses were performed using Statistical Package for Social Science; Version 7.5 for Windows 95). P < 0.05 was considered significant. Multiple regression analyses were performed with cord arterial lipid peroxide and acid-base values as dependent variables and mode of delivery, parity, gestational age, duration of labour, birthweight and body mass index as independent variables to assess the independent contribution of the labour process.
The study was approved by the Human Research Ethics Committee of the Chinese University, and written informed consent was obtained from all women who participated.
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Of 112 paired blood samples from elective caesarean deliveries initially studied, 21 (19%) had acid-base parameters which were not sufficiently different from one another to confirm separate arterial and venous origins. These cases were excluded from analysis, leaving 91 cases for cord arterial lipid peroxide assay. Of these 91 elective caesarean deliveries, nine had gestational diabetes and seven had other mild medical disorders: genital warts (n = 2); asthma (n = 2); essential hypertension (n = 1); rheumatoid arthritis (n = 1); and neurofibromatosis (n = 1). There were no significant differences in either malondialdehyde or organic hydroperoxides between women with or without medical conditions. The indications for caesarean section in the remainder were breech presentation (n = 15), placenta praevia (n = 16) or repeat caesarean section (n = 44). In 77 women the operation was performed under epidural anaesthesia, and in 14 under general anaesthesia. There was no significant difference in cord arterial pH, pCO2 organic hydroperoxides or malondialdehyde between those receiving general anaesthesia and those operated on under epidural blockade. Both PO2 and base excess were significantly lower in the epidural group in our study (P < 0.05).
These 91 cases were compared with 182 normal vaginal deliveries with confirmed arterial samples assayed for lipid peroxidation (152 of these were reported in an earlier paper4). Women undergoing caesarean section were older (P < 0.05), shorter (P < 0.01), and of lower gestational age (P < 0.001) than those with normal vaginal deliveries and their babies were lighter (P < 0.001). (Table 1)
Table 1. Maternal obstetric demographic characteristics. Values are given as mean (SD) [range]. CS = caesarean section; SVD = spontaneous vaginal delivery.
| ||Elective CS (n = 91)||SVD (n = 182)|
|Weight (kg)||65.21 (9.43)||65.51 (9.14)|
|Gestational maturity (weeks)||37.89(3.87)||39.74(1.22)|
|Birthweight (kg)||2.98 (0.56)||3.26 (0.40)|
Table 2 shows a comparison of the cord arterial malondialdehyde organic hydroperoxides, pH, pCO2 PO2 and base excess levels between 91 elective caesarean deliveries and 182 normal vaginal deliveries, using the Mann-Whitney U test; the 10th, 50th and 90th centiles are shown for each group. Table 3 shows a cross-tabulation of outcome measures in terms of low pH and base excess (< 10th centile for vaginal deliveries) compared with high levels of lipid peroxidation (> 90th centile for vaginal deliveries).
Table 2. Comparison of cord arterial lipid peroxide concentrations and acid-base balance according to mode of delivery. Values are given as median with 10th and 90th centile values. CS = caesarean section; SVD = spontaneous vaginal delivery; OHP = organic hydroperoxide; MDA = malondialdehyde; BE = base excess.
| ||Elective CS (n = 91)||SVD (n = 182)|| |
| ||10th centile||Median||90th centile||10th centile||Median||90th centile||P|
Table 3. Comparison of neonatal outcome in terms of cord acid-base balance and levels of lipid peroxidation. Numbers of incidence are shown. Key as for Table 2.
| ||Outcome: acid-base balance*|
| ||pH & BE normal||pH < 7.14 or BE < −9.77||pH < 7.14 & BE < −9.77|
|Outcome: lipid peroxidation**|
| MDA & OHP normal range||134||6||11|
| MDA>1.62 or OHP>0.25||20||2||4|
|Elective caesarean delivery|
| MDA & OHP normal range||85||2||—|
| MDA > 1.62 or OHP > 0.25||4|| ||—|
| MDA > 1.62 & OHP >0.25||—||—||—|
Cord arterial plasma levels of malondialdehyde and organic hydroperoxides were significantly higher in the women who had undergone labour (Mann-Whitney: P < 0.001) than those who underwent elective caesarean delivery. Base excess and pH, but not pCO2 and PO2 were also significantly lower amongst vaginal deliveries. Figure 1 shows a graph of malondialdehyde plotted against pH for the two delivery groups, and clearly demonstrates the higher malondialdehyde levels associated with labour. Figures 2–4 show similar plots for OHP and pH, malondialdehyde and base excess, and organic hydroperoxides with base excess.
Figure 1. Scatter plot of cord arterial malondialdehyde (MDA) concentration (μmol/L) against pH with cases labelled according to mode of delivery. ▴= elective caesarean section; ⊡= normal vaginal delivery.
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Figure 2. Scatter plot of cords arterial organic hydroperoxide (OHP) (μmol/L) against pH with cases labelled according to mode of delivery ▴= elective caesarean section; ⊡= normal vaginal delivery.
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Figure 3. Scatter-plot of cord arterial malondialdehyde (MDA) concentration (μmol/L) against base excess (mmol/L) with cases labelled according to mode of delivery. ▴= elective caesarean section; ⊡= normal vaginal delivery.
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Figure 4. Scatter-plot of cord arterial organic hydroperoxide (OHP) (mmol/L) against base excess (mmol/L) with cases labelled according to mode of delivery. ▴= elective caesarean section; ⊡= normal vaginal delivery.
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Multiple regression analyses demonstrated that the effect of mode of delivery (labour) on malondialdehyde, organic hydroperoxides, pH and base excess was statistically independent (P < 0.001) of effects due to maternal age, parity and body mass index, gestational age and birthweight. Parity was the only other significant independent variable (P < 0.05).
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Meaningful audit of obstetric practice requires parameters that can be measured objectively and which confirm clinically defined perinatal asphyxia11,12 Currently determination of umbilical cord blood acid-base status provides the only widely accepted biochemical assessment of perinatal asphyxia9. Both pH and base excess however, only reflect fetal metabolic adjustments and therefore are not directly related to fetal damage, except where extreme values exist13. We therefore suggest that umbilical cord lipid peroxide concentrations could offer a more appropriate outcome measure as they reflect the extent of cell membrane damage by free oxygen radicals14,15.
All the women studied were at term with no evidence of fetal compromise at recruitment. The anaesthetic technique used during elective caesarean delivery has been shown to affect umbilical pH and PO2, with epidural anaesthesia having the least effect16. Epidural anaesthesia was used in most of our cases and the mean arterial pH levels were similar to those reported elsewhere. Both PO2 and base excess were significantly lower in the epidural group in our study (P < 0.05), although all values were within the 10th and 90th centiles for vaginal delivery. There were no significant differences in pH, PCO2, organic hydroperoxides or malondialdehyde between women delivered by elective caesarean section under general or epidural anaesthesia. Conversely, epidural anaesthesia in labour has been shown to be associated with lower levels of lipid peroxidation due to a reduction in the effect of prolonged second stage.13 However, all the vaginal deliveries reported here had second stage lengths less than 40 minutes so any effect of epidural anaesthesia is minimised in this study.
The use of women having elective caesarean section at term under conduction anaesthesia to reflect the normal fetal milieu has been criticised by Gregg and Weiner17 on the basis that umbilical blood gas measurements are significantly different from those obtained by cordocentesis at term. However, they did not perform paired tests in their study, so selection bias may be responsible for their findings. Women having elective caesarean section nevertheless provide a convenient baseline for clinical measurements, especially where comparison with labouring women is required. We compared these with normal vaginal deliveries as, with a few exceptions, these represent the normal fetus undergoing a normal physiological stress, leading to a good neonatal outcome: In our study only four infants (2.2%) in the normal vaginal delivery group exhibited signs of fetal distress; all other cases of fetal distress underwent caesarean or operative vaginal delivery and are therefore not reported here.
The significant differences in lipid peroxidation between elective caesarian deliveries and those following labour relate mostly to intrapartum events; the multiple regression analysis demonstrated that the effects of labour on both malondialdehyde and organic hydroperoxides (and on pH and base excess) were independent of maternal age and body mass index, of gestational age, overall labouring duration and birthweight. Uterine contractions during labour result in diminution of the arterial inflow to, and cessation of, venous drainage from the placenta. This situation is reversed during the relaxation phase, leading to recurrent ischaemia-reperfusion, free radical production and a rise in lipid peroxidation. Organic hydroperoxides levels, although significantly lower in elective caesarean section compared with vaginal deliveries, did show considerable overlap in values. Malondialdehyde levels were significantly lower in infants born by elective caesarean relative to those born by the normal vaginal route, although there was still minor overlap (nine samples ≤ 10th centile). This probably reflects the higher levels of prostaglandin activity in labouring women. Unlike organic hydroperoxides, the amount of MDA measured by the TBARS (thiobarbituric acid-reactive substances) reaction incorporates both circulating malondialdehyde and that derived from breakdown of both organic hydroperoxides and endoperoxides during the assay technique18. The relation between plasma malondialdehyde and organic hydroperoxides is a dynamic one: organic hydroperoxides rising initially and then declining whilst malondialdehyde, which is breakdown product of lipid peroxidation, rises. This dynamic process is further complicated in the fetus by placental removal of malondialdehyde 7, which is a highly toxic chemical due to its ability to form disulphide bridges across nucleotide or amino acid chains.
At the present time we are unable to determine what concentration of lipid peroxides is indicative of permanent (irreversible) neonatal damage, as all the infants in these studies ultimately had a satisfactory outcome. At this stage we must assume that the levels of lipid peroxidation observed in the labour group indicate cellular damage that is within the scope of the neonate to repair without significant long term sequelae. The low levels of peroxidation associated with elective caesarean section should not therefore be used to construe any benefit associated with this approach at present. Nevertheless, the measurement of lipid peroxidation products provides substantive evidence that free oxygen radical activity is markedly increased during the process of labour, and that this has some deleterious effects at the cellular level, in which lipid peroxides decrease membrane fluidity, inactivate membrane-bound receptors and enzymes, and to increase the ‘leakiness’ of the membrane to substances that do not normally cross it (such as calcium ions)19. As the fetal brain is protected from the effects of mild to moderate hypoxia by redistribution of oxygenated blood flow away from nonvital centers, it is unlikely that any of the lipid peroxidation observed in these studies arises from neural damage. However, clinical studies have shown that malondialdehyde and organic hydroperoxides concentrations provide a good measure of fetal hypoxic insult during labour, rising in situations known to be associated with hypoxic morbidity and such high concentrations being preventable by appropriate obstetric intervention in these situations.
In conclusion, the findings of our study support the proposed model of cellular damage by free radical activity following recurrent hypoxia-reperfusion during labour, but do not, as yet, allow definition of morbid concentrations of lipid peroxidation in cord arterial blood.