Objective
The aim of this study was to assess the involvement of lipoxygenase (LOX) metabolic pathways in uterine tissues from pregnant women as well as the combined inhibition of LOX and cyclooxygenase (COX) on contractile activity.
Study Design
Uterine biopsies were performed from consenting women undergoing elective caesarean sections at term (n = 24). Western blot analysis and isometric tension measurements were performed in vitro on fresh human myometrial strips. Concentration-response curves to arachidonic acid (AA) 861 and baicalein (5- and 12-LOX inhibitors, respectively) were performed. The combined effects of baicalein and indomethacin were also assessed. Contractile activities were quantified by calculating both amplitude and the area under the curve over 20 minute periods.
Results
5- and 12-LOX were present in all tested tissues. Addition of AA861 or baicalein resulted in tocolytic effects ( P < .05). Finally, the combined inhibition of both COX and 12-LOX pathways resulted in additive tocolytic effects.
Conclusion
5- and 12-LOX pathways modulate human myometrium contractility.
Preterm birth is a growing problem in all developed countries, with spontaneous preterm birth representing half of the cases. The most important treatment for preterm labor is tocolytic drugs, although their efficacy is very limited. Current treatments can prolong pregnancy for only no more than 48 hours.
Numerous studies have assessed the role of arachidonic acid (AA) metabolites on myometrium contractile activity during pregnancy. At the present time, it is well known that prostaglandins (PGs) have important effects on the contractile activity of the mammalian myometrium as well as a key role in parturition. In 2004 indomethacin, a cyclooxygenase (COX) inhibitor, was considered as the most commonly used drug for the treatment of preterm labor in Canada.
A second pathway has also been recently described within the AA metabolic pathway, namely the epoxy-eicosanoid pathway. Indeed, studies have demonstrated the presence of the CYP-450 enzymes that produces epoxyeicosatrienoic acids (EETs) and 20- hydroxyeicosatetraenoic acids (HETE) in the myometrium, which have been depicted as having putative tocolytic properties. Finally, a third pathway involves the leukotriene branch of arachidonic acid metabolism, as illustrated in Figure 1 .
Leukotrienes are synthesized by stepwise chemical modifications of AA by the action of lipoxygenases (LOX). The presence of the 5-, 12-, and 15-LOX isoforms has been detected in uterine tissue at different stages of pregnancy in baboons and humans. The human amnion, chorion, deciduas, and placenta synthesize various compounds such as HETEs and leukotrienes (LTs), which may be involved in the onset of labor. Because LTs (LTC 4 , LTD 4 ) increase uterine contractile activity, it has been proposed that LOX inhibitors could display tocolytic effects and consequently be of potential interest as pharmacological agents in the treatment of preterm labor.
However, no study has addressed the effects of LOX inhibitors on human uterine contractile activity. The aim of this present work was to investigate the involvement of LT metabolic pathways in uterine tissues from pregnant women as well as the effect of the combined addition of COX and LOX inhibitors on spontaneous uterine contractile activity.
Materials and Methods
Study population
Patients admitted for an elective cesarean section were asked to participate in the study. All patients had a low transverse hysterotomy incision. The study was approved by our institutional Ethics Committee for research on human subjects (project #09-040; clinicaltrials.gov identifier NCT00939744 ), and all volunteers gave written informed consent.
The inclusion criteria were: (1) a gestational age between 37 0/7 and 40 0/7 weeks of gestation, (2) a singleton gestation, (3) no labor, and (4) a signed informed consent. Women were excluded if presence of infections (chorioamnionitis, human immunodeficiency virus, genital herpes, hepatitis B and C) or vaginal bleeding after the third trimester was detected. Medical data were obtained from the patients’ medical files.
Sample collection
During the cesarean section, immediately after delivery of the baby but prior to maternal injection of oxytocin, all biopsies of myometrium were excised from the upper lip of the lower uterine segment incision in the midline. Placenta biopsies (1 cm 3 ) were sampled immediately after removal in periphery on the maternal face, and the membranes were sampled free of placenta in a systematic manner by the same investigator.
Once collected, all tissue biopsies were placed in Krebs-Heinseleit physiological salt solution (Krebs) of the following composition (millimoles per liter): 4.7 potassium chloride, 118 sodium chloride, 1.2 magnesium sulfate, 2.5 calcium chloride, 1.2 potassium phosphate, 25 sodium bicarbonate, and 11.1 glucose (Sigma-Aldrich, St Louis, MO) at pH 7.4. Tissues were stored at 4°C and used within 24 hours of collection or were rapidly rinsed before snap freezing in liquid N 2 and subsequently stored at –80°C until analysis.
Western blot analysis
Subcellular fractions (cytosolic and microsomal) were prepared from myometrium, fetal membranes, and placenta and separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis as previously described. For Western blot analysis, membranes were blocked for 2 hours with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween at room temperature. Blots were incubated overnight at 4°C with rabbit antiserum raised against either 5- or 12-LOX proteins (Abcam, Cambridge, MA). After washing, the membranes were incubated in a solution containing peroxidase-conjugated donkey antirabbit immunoglobulin G antiserum (Amersham, QC, Canada). Enhanced chemiluminescence kit (Roche, QC, Canada) was used to detect protein labeling.
Isolated organ bath experiments
Longitudinal myometrial strips (measuring approximately 2 × 2 × 10 mm) were dissected, cleansed of adherent myometrial tissue, and mounted for isometric recording under 2 g of resting tension in an organ bath system as previously described. The tissue baths contained 10 mL of Krebs solution maintained at 37°C, pH 7.4, and were continuously gassed with a mixture of 95% oxygen/5% carbon dioxide.
Myometrial strips were allowed to equilibrate for at least 2 hours, after which a 30 minute period was allotted to achieve spontaneous phasic contractions. Passive and active tensions were assessed using transducer systems (Radnoti Glass Technology, Monrovia, CA) coupled to Polyview software (Grass-Astro-Med Inc, West Warwick, RI) for facilitating data acquisition and analysis.
Drugs and chemical reagents
AA861 (Sigma-Aldrich), baicalein (Cayman Chemical, Ann Arbor, MI), and indomethacin (Cayman Chemical) were dissolved in 100% ethanol (EtOH) and stored as 10 mM stock solutions. The final bath concentration of EtOH never exceeded 0.3%. Exogenous inhibitors were added separately to the tissue bath or in a cumulative manner at increasing concentrations (10 nM to 10 μM) in 30 minute intervals.
Two sets of control experiments were performed as follows: in control 1 (time), strips were exposed to Krebs solution only for up to 6 hours; in control 2, strips were exposed to Krebs solution and vehicle (EtOH). Fresh Krebs solution was prepared daily.
Data analysis and statistics
The amplitude and the area under the curves (AUCs) for the isometric tension studies were calculated on raw recordings from independent myometrial strips. The effect of pharmacological agents and respective controls were assessed by calculation of the AUC for each 30 minute interval.
Values were normalized as a percentage of the AUC obtained in the 30 minute basal activity period and were corrected for the reduction in contractile activity observed with the vehicle (control 2) such that the provided mean maximum inhibition values (MMIs) represented the net inhibition. Contractile activities were quantified by calculating the amplitude and the AUC for each experimental condition using Sigma Plot 11.0 (SPSS Inc, Chicago, IL).
Data were not normally distributed and were therefore analyzed with nonparametric tests. The Wilcoxon signed rank test was used for paired results and the Mann-Whitney test for unpaired results. Differences were considered significant when P < .05.
Results
Study population
The study group comprised 24 healthy white pregnant women with a mean age of 30.2 years (range, 19–44 years; Table 1 ). Participants underwent cesarean delivery between 37 0/7 and 40 0/7 weeks of gestation (mean age, 38 6/7 weeks of gestation) with a mean body mass index (BMI) of 26.5 kg/m 2 (range, 17–47 kg/m 2 ). Indications for a cesarean section included previous cesarean sections (n = 12), placenta previa (n = 3), and breech position (n = 9). The majority of pregnant women were nonsmoking (83%).
| Variable | Number of patients (n = 24) |
|---|---|
| Maternal age, y | |
| <20 | 1 (4.16%) |
| 20–35 | 18 (75.0%) |
| ≥35 | 5 (20.8%) |
| Ethnicity | Caucasian (100%) |
| Parity | |
| Nulliparous | 4 (16.7%) |
| Multiparous | 20 (83.7%) |
| BMI, kg/m 2 | |
| <20 | 3 (12.5%) |
| 20–25 | 8 (33.3%) |
| >25 | 13 (54.16%) |
| Indications for cesarean section | |
| Pregnancy complications a | 3 (12.5%) |
| Breech presentation | 9 (37.5%) |
| Previous cesarean section | 12 (50%) |
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