Nitric oxide (NO), a unique and ubiquitous biological messenger, may have important roles in physiological and pathophysiological processes during pregnancy , including maintaining uterine quiescence  and modulating blood flow to uterus and fetal organs . More importantly, in the absence of autonomic innervation , vascular tone in the fetoplacental circulation is dependent on endocrine, paracrine and circulating vasoactive factors and NO is one of the factors modulating fetoplacental blood flow .
NO is synthesized by nitric oxide synthases (NOS). NOS consists of three isoforms known as endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS) . It has been reported that numerous factors including acetylcholine, glutamate, physical pressure and shear stress may affect the functional expression of NOS . It was found that citrulline (a marker for NO production) level in cerebrospinal fluid (CSF) was significantly decreased in parturients experiencing labour pain during active labour compared with control women without labour pain subjected to elective Caesarean section  and that plasma concentrations of NO metabolites, i.e. nitrate and nitrite, were significantly lower in women with labour pain in active labour than pregnant women without active labour in the third trimester of pregnancy . These findings indicate that the production of NO is suppressed during active labour probably by labour pain; however, Hsu and colleagues  revealed no differences in CSF concentrations of NO metabolites between labouring and non-labouring women.
Since the levels of NO metabolites appear to be lowered during active labour and the underlying mechanisms remain unknown, we hypothesized that labour pain may play a role in modulating NO production and that combined spinal-epidural analgesia (CSEA) that relieves labour pain effectively may affect circulating levels of NO metabolites. To test this hypothesis, we determined the serum concentrations of NO metabolites, i.e. nitrate and nitrite, in pregnant women during labour with or without using CSEA that is one of the most commonly used for labour pain relief .
The Institutional Review Board (IRB) of Zhejiang University School of Medicine approved the protocol of the current investigation and all participants provided their written informed consents. Fifty pregnant women were enrolled in the study when their cervical dilation was 3˜4 cm. All women met the following criteria: Chinese, nulliparous, singleton pregnancy, spontaneous onset of labour, vertex, 36-41 weeks of gestation, ASA physical status I or II. On their arrival at the delivery unit, all women were informed of the availability of pain relief including CSEA and were allowed to ask for pain relief. Women who had pregnancy complications, fetal abnormalities or received systemic anaesthetics or antalgics during labour were excluded.
Twenty-five women who met the inclusion criteria and requested CSEA were recruited in the CSEA group. Consecutive 25 pregnant women who did not request CSEA and were matched for maternal age, gestational age and the stage of cervical dilatation served as control. Control women did not receive any other analgesia.
Combined spinal-epidural analgesia
In the CSEA group, a ‘needle through needle' technique (using a CSEA-set, B-D, China) for CSEA was used with the women in the right lateral position. After a 17-G Tuohy needle was inserted into the epidural space using the method of loss of resistance to air at the L2˜3 interspace, a 25-G Whiteacre spinal needle was passed through the epidural needle into the subarachnoid space. When the flow of cerebral spinal fluid was confirmed, 3.75 mg of ropivacaine (Naropin, AstraZeneca, Sweden) and 25 μg of fentanyl (Fentaini, Renfu, China) in a volume of 2.5 mL were injected and then the spinal needle was removed. An epidural catheter was advanced 3 cm into the epidural space and the women were then moved to supine position with left uterine displacement. A continuous infusion pump (Bexter II, Bexter, USA) was connected to the epidural catheter and appropriate settings for later use were entered. No epidural drug was given until discomfort returned, when patients received 15 mL of 0.2% ropivacaine in divided doses to achieve a bilateral T10 sensory level, and an infusion of 0.1% ropivacaine with 2 μg·mL−1 fentayl was started at 10 mL h−1. The analgesic effect, motor blockade, breathing were monitored by an anesthesiologist and confirmed by another independent anesthesiologist.
Blood pressure (BP), heart rate (HR) and visual analogue pain scores (0-10 cm scale, 0 = no pain and 10 = worst possible pain) were monitored or evaluated at study entry. Study entry was set when the parturients had cervical dilation of 3˜4 cm and were enrolled into the present study. They were evaluated at 5-min intervals after study entry (after CSEA in CSEA group) for 30 min and at 20-min intervals until baby delivery. Labour progress was independently monitored and managed by our obstetricians. Cervical examination was performed in a regular interval of 2 h during latent phase and 1 h during active phase. Fetal HR and uterine contractility (external tocodynamometry, ET) were recorded continuously using electronic FHR&ET monitor. Adverse effects of CSEA such as postdural headache, pruritus as well as hypotension, the administration of oxytocin as well as i.v. Ringer's Solution and the artificial rupture of membrane were recorded.
In both groups, blood samples were taken at study entry (specimen 1), full cervical dilation (specimen 2) and 2 h after delivery of the baby (specimen 3). Sera were collected, aliquoted and stored at −80°C until assay.
NO was determined by the evaluation of its oxidation products (nitrites and nitrates) where the nitrates were reduced to nitrite; the total concentration of nitrite was measured by using Griess reaction as we previously described .
Continuous data were normally distributed as tested by the Kolmogorov-Smirnov test and presented in mean ± SD. The t-test or one-way ANOVA was used to compare means where appropriate and the χ2 test for incidence data. SPSS 11.5 (SPSS Inc., Chicago, IL, USA) was used for data analysis. P < 0.05 was considered statistically significant.
Of the 50 women, 11 withdrew because they required Caesarean section due to the abnormalities of fetal HR or obstructed labour. Of these, 5 belonged to the CSEA group and 6 to the control group. This left 20 women in the CSEA group and 19 in the control group who finished the investigation and were included in data analysis.
There were no significant differences in maternal age, gestational age, height, weight and cervical dilation at study entry (P > 0.05 for all, Table 1). Although there were no significant differences in the pain scores at the study entry (P > 0.05, Table 2), they were significantly lowered after analgesia in the CSEA group and was significantly lower in the CSEA group than in the control group after analgesia (P < 0.05). Before labour (as measured at last prenatal care) and at study entry, mean BPs were not significantly different between the groups (P > 0.05 for both). In the control group, mean BP was significantly increased at study entry and during labour compared with pre-labour (P < 0.05 for all). After analgesia, mean BP was significantly lower in the CSEA group than in the control group (P < 0.05 for all). At 6˜7 cm cervical dilation, the uterine contractility was significantly stronger in the CSEA group than in the control group (P < 0.05, Table 2).
The duration of labour and labour management are shown in Table 3. The active phase in the first stage of labour was significantly shorter but second stage was significantly longer in the CSEA group than in the control group (P < 0.05 for both). There was no significant difference in Ringer's solution infusion during study period, the oxytocin maximum infusion rate, the rate of artificial membrane rupture and the mode of delivery (P > 0.05). Headache and hypotension were not found but pruritus was observed in five cases.
Serum concentrations of NO metabolites are shown in Table 4 and Figure 1. There was no significant difference in serum concentrations of NO metabolites at study entry (P > 0.05). In the control group, serum levels of NO metabolites were significantly decreased at full cervical dilation and 2 h post partum compared with those of study entry (P < 0.05 for both). In the CSEA group, there were no significant differences in serum concentration of NO metabolites during and after labour (P > 0.05). Serum concentrations of NO metabolites were significantly lower in the CSEA group than in the control group at full cervical dilation and at 2 h after delivery of baby (P < 0.05 for both).
In the present study, we showed that the administration of CSEA resulted in lower visual analogue scale value, shorter active phase of labour and stronger uterine contraction as well as lack of increase in mean BP compared with control. More importantly, we demonstrated that maternal serum levels of NO metabolites decreased significantly during active labour in the control group and this decrease was almost abolished in the CSEA group. Our findings indicate that the decrease in the levels of NO metabolites during active labour may be associated with labour pain, and CSEA abolishes the alteration in NO metabolites. To the best of our knowledge, this is the first report regarding the effect of CSEA on NO metabolites.
The levels of NO metabolites have been reported as being changed during pregnancy and parturition. Jo and colleagues  reported maternal serum levels of nitrite and nitrate in normal pregnancy were decreased near term and further decreased during labour. Yanik and colleagues  and Chen and colleagues  found that serum levels of NO metabolites were significantly decreased during the labour and were lower at active phase than at latent phase, which was in agreement with our findings in control group. However, the exact mechanisms by which NO production was decreased as labour progresses have not been clearly clarified yet. Nanno and colleagues  found that the plasma concentrations of NO metabolites decreased significantly at vaginal delivery but not at Caesarean delivery, which was independent of the plasma artrial natriuretic peptide and brain natriuretic peptide concentrations, suggesting the possible transient down-regulation of maternal NOS during parturition.
The effect of CSEA on the levels of NO metabolites has never been reported yet. In the current study, the administration of CSEA led to the relief of labour pain and avoided the decrease in serum NO metabolites, implying possible link between relief of labour pain and alteration of NO production. However, the mechanisms behind remained unclear. It was evidenced that serum levels of catecholamines, epinephrine and norepinephrine, were increased during painful labour and that effective labour pain relief decreased circulating catecholamines , while catecholamines are able to block NO production by microphage . Serum adrenocorticotrophin (ACTH) and glucocorticoids were increased during labour probably because of labour pain and stress  and epidural labour analgesia attenuated the rising of maternal serum ACTH and glucocorticoids , while Wallerath and colleagues [20,21] reported that glucocorticoids such as dexamethasone down-regulated the expression of eNOS by human endothelial cells. Spinal anaesthesia produces partial sympathetic block, which may result in the vasodilation in the blocked area of the body , increase in blood flow, improvement of the function of endothelial cells and activity of eNOS. Very recently, Gonca and colleagues  found that the expression of iNOS was up-regulated during epidural analgesia. These mechanisms may partly explain why CSEA attenuated increase in serum concentration of NO during labour. However, we could not make a clear conclusion that the change of serum levels of NO metabolites due to CSEA was the effect of labour pain relief itself or a consequence of reduced BP or the change in circulating neuro-hormones resulted from labour pain relief. Different isoforms of NOS have their different localization, regulation, catalytic properties and inhibitor sensitivity . Stress and hypoxia are known to induce NOS activity . However, we could not tell the exact NOS isoform which is the dominant one involved in the NO production during CSEA.
NO production may be affected by the state of blood flow and BP. Woodman and colleagues  revealed that increased blood vessels' shear stress induces eNOS mRNA expression and improves endothelium-dependent dilation in senescent soleus muscle feed arteries (SFA) by increasing NO production. And Lyamina and colleagues  also found that there was a correlation between hypertension and NO production. These findings suggest that high blood flow and hypertension may induce the production of NO. In present study, BP was higher in the control group with labour pain compared to CSEA group, whereas the serum levels of NO metabolites were lower in the CSEA group than in the control group. Our findings suggest the decrease in BP induced by CSEA may be one of the factors regulating NO production during labour. Given that there was no difference in the amount of liquid infusion between the groups, the liquid infusion did not affect the results about serum levels of NO metabolites in the present study.
Metabolites of NO are cleared by kidneys and the clearance is influenced by renal blood flow (RBF) and glomerular filtration rate (GFR). Epidural or spinal anaesthesia blocks the sympathetic nervous fibers to the kidneys originating from the lower thoracic segment of the spinal cord (T7-T11) and increases RBF and GFR, implying that CSEA may lead to the decrease of NO metabolites in serum, which is contrast to the increase of serum NO in the current study. However, a recent study found that epidural anaesthesia did not alter the RBF in healthy people , suggesting nitrate/nitrite concentration be not affected by epidural or spinal anaesthesia. During CSEA used for labour analgesia, the administration of anaesthetics is localized and the dose is low, we believe that anaesthetics will not affect NO production or metabolism in such a situation, although no study has been available in this regard.
Consistent with previous reports, CSEA provided effective analgesia during active labour in the current investigation. Dresner and colleagues . reported that the administration of CSEA during labour made 74% of parturients feel ‘excellent' and 61% feel ‘satisfactory'. Recently, Moschini and colleagues  found that 73% of parturients felt ‘excellent' and 20% parturients felt ‘good' during CSEA labour analgesia. Tsen and colleagues  observed that CSEA led to more rapid cervical dilation compared with epidural analgesia, while we found that the duration of active phase was shortened in CSEA group. These findings indicate that combined spinal epidural analgesia is effective in relieving labour pain and accelerating labour process.
Our experiment design followed previous report . The bias in selecting subject could not be excluded due to the lack of randomization and we could not group randomly the pregnant women into CSEA or control group because of the ethical considerations. However, there were no differences in serum levels of NO metabolites and cervical dilatation at study entry, as well as maternal age, gestational age and parity between the groups, indicating the subjects in the CSEA and control groups were quite comparable.
To summarize, CSEA relieved labour pain effectively, attenuated the increase in mean BP, strengthened uterine contraction, shortened the duration of active labour, meanwhile it abolished the decrease of NO production during labour, suggesting that NO may be one of the mediators of these CSEA effects and that it may be related to labour pain.
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