The Effects Of Elective Cesarean Section On Airway Epithelial Sodium Channels And Pulmonary Surfactant Homeostasis In Term Infants

During the past ten years, some pregnancy women have preferred elective cesarean section (ECS) over normal vaginal delivery (VD). ECS is also termed as planned cesarean section and executed prior to labor. It refers to a cesarean section that is performed on a pregnant woman on the basis of an obstetrical or medical indication or at the request of the pregnant woman. Some pregnancy women believe that ECS may keep them from pain during the course of delivery. However, it is found that neonates, who are delivered by ECS, would be suffered by respiratory disorders and even get sever respiratory failure after birth. It is speculated that infants delivered by ECS would not adapt to the condition outside as soon as possible. These infants would more likely be admitted to neonatal intensive care unit (NICU) and received additional medical interventions, such as oxygen therapy, artificial ventilation support. The rate of cesarean section in China has been at a relatively higher level (The mean rate may be near to50%, even much higher somewhere.), when compared with that of other countries, although Government has tried to decrease the level. Most patients and health care providers who prefer cesarean section over vaginal delivery always cite as reasons for their preference, not only the greater control and convenience that cesarean section promises, but also less fears associated with vaginal delivery (such as perineal injury, including anal and urinary incontinence, sexual dysfunction). More and more evidences are shown that ECS would be related to some respiratory morbidities, such as transient tachypnea of newborn (TTN), respiratory distress syndrome (RDS) and persistent pulmonary hypertension of newborn (PPHN). The mechanisms, on which some respiratory disorders are caused by ECS, are not really understood and need further study.TTN is a common respiratory disorder, which always happens to term or near term infants. Lack of labor during ECS was thought to be the main reason to TTN in the past. Amiloride sensitive epithelial sodium channels (ENaC) emerge as a key event in the transepithelial movement of alveolar fluid now. It is shown that in utero, the entire airway epithelium is likely to be under a homogeneous humoral and local ontogenic regulation. It was also demonstrated by previous data, that the function of nasal epithelial is well correlated with some neonatal lung diseases, and nasal epithelial can be used as a surrogate for lung epithelium. To our knowledge, there were no consistent results on the relationship between delivered mode and the expression of ENaC in pervious literature.RDS is another common respiratory disorder, which often happens to preterm infants. However, more and more cases of term infants with RDS have been shown during these years. Lack of pulmonary surfactant (PS) is still thought as the main cause of RDS. Lamellar bodies (LB) are packages of surfactant, which is secreted into alveolar spaces between24and26weeks'gestation or earlier. The lamellar bodies count (LBC) in the amniotic fluid, reflecting surfactant production and release, has gained popularity as a cheap and easily accessible test of fetal lung maturity, because prior tests involving phospholipids analysis are expensive, labor intensive., and unavailable in many clinical settings. LBs are similar in size to platelets, thus LBC could be determined quickly with a conventional hematology analyzer. There are a few studies, focusing the issue of the relationship between LBC and delivery mode, but the results are still not consistent. ABCA3, a member of the ATP binding cassette (ABC) transporter superfamily, is expressed exclusively in alveolar type Ⅱ cells and is localized mostly at the limiting membrane of the lamellar bodies in human. It is also found that the level of ABCA3protein is dramatically increased during the last trimester of pregnancy, and functions as a transmembrane transporter of lipid components of pulmonary surfactant. Recently, various mutations in the ABCA3gene have been identified in term newborns with RDS and in children with interstitial lung disease, also indicating the important role of ABCA3in human lung disease. To our knowledge, there was no study on the relationship between the expression of ABCA3and delivery mode in previous studies.In our present study, firstly, we retrospectively studied near20,000infant during the past two and a half years, and explored the relationship between ECS and early respiratory morbidities. Secondly, we addressed the difference in the expressions of ENaC between term infants delivered by ECS and those by VD. Thirdly, we also compared the difference in LBC between term infants delivered by ECS and those by VD. Finally, we analyzed the different in the expression of ABCA3, which is mainly located at the membrane of LB, between the term infants delivered by ECS and those by VD.Part one:The effects of elective cesarean section on early respiratory morbidities in term infants:a retrospective review.Aim:1. To review the information about a total of20000neonates and describe the characteristics of the data. 2. To compare the respiratory morbidities between infants delivered by elective cesarean section and those by vaginal delivery.Methods:1. We collected and reviewed the information of all term infants delivered by ECS or VD during the year2006-20082. We compared the early respiratory morbidities between infants delivered by ECS and those by VD.3. We analyzed whether ECS was an independent risk factor to some early respiratory morbidity by the mode of binary Logistic Regression.Results:1. The cohort study comprised19123term infants, among which64.4%(12310/19123) infants were delivered by ECS,35.6%(6813/19123) infants were delivered by VD;51.7%infants were male;82.1%born at38-40gestational age;91.2%born with normal birth body weight (2500-4000grams). The mean age of mother in our study was28.8±3.6years,90.0%mothers were primipara,69.6%mothers lived in city, and about9%mothers were with pregnancy complications.2. The infants who were delivered by ECS, were with higher birth weight, lower gestational age, higher morbidity of TTN and RDS, and more admission rate of NICU, but higher Apgar score at1min, lower morbidity of birth trauma, and lower morbidity of hypoxic ischemic encephalopathy (HIE), when compared with those delivered by VD (P<0.05, respectively). Furthermore, the mothers who preferred to ECS, were older and with higher morbidities of pregnancy complications, when compared with those preferred to VD (P<0.05).3. ECS, lower birth weight, and Apgar score<7at1min, were independent risk factors to morbidity of TTN in term infants (P<0.05). However, we did not found the independent risk factors to morbidity of RDS in term infants (P>0.05). The possibility of ECS, as an independent risk factor to RDS morbidity, was at the level of0.051.Conclusions:1. ECS was the main delivery mode in our hospital during the period of study.2. ECS was an independent risk factor to the morbidity of TTN.3. ECS may be associated with the morbidity of RDS.Part two:The effects of elective cesarean section on the expression of epithelial sodium channels of air way in term infantsAim:1. To investigate the effects of ECS on the expression of all epithelial sodium channels (ENaC) subunits mRNA in term infants.2. To investigate the effects of ECS on the protein level of ENaC subunits in term infants.Methods:Clinical prospective cohort study1. We collected the samples of nasal epithelial cells from the term infants delivered by ECS or VD within15-30min after birth.2. We identified the expressions of all ENaC subunits mRNA between term infants delivered by ECS and those by VD by real-time PCR.3. We identify the protein levels of all ENaC subunits between term infants delivered by ESC and those by VD by Western blot. Results:1. The cohort study comprised306infants, half of whom were delivered by ECS. There were2infants with TTN, who were delivered by ECS, then admitted to NICU. No infants with RDS were found during the study period.2. The mean mRNA expressions of ENaC a,(3and y in the infants delivered by ECS were lower than those in infants delivered by VD (0.041±0.080versus0.056±0.039, P<0.05,0.010±0.020versus0.016±0.014, P<0.01, and0.021±0.018versus0.026±0.025, P<0.05, respectively).3. Regardless of delivery mode, the mean mRNA expressions of ENaC α and β in the infants born at37weeks gestational age were lower than those with>37weeks gestational age. However the mean mRNA expression of ENaC y was similar in term infants with different gestational age.4. The mean levels of ENaC β and γ subunits were lower in the infants delivered by ECS, when compared with those in the infants delivered by VD (1.00±0.2versus0.30±0.30,P<0.01and1.00±0.1versus0.50±0.06, P<0.05), However, the mean level of ENaC a subunit was similar between the infants delivered by ECS and VD (1.00±0.05versus1.03±0.12, P>0.05).Conclusions:1. The expressions of all ENaC subunits were decreased in infants delivered by ECS, compared with those in infants delivered by VD.2. Regardless of delivery mode, the changes in the expression of α and β ENaC subunits mRNA was in a timely regulation manner according to gestational age.3. The change of ENaC β and γ subunits protein levels may be more susceptible by delivery mode. Part three:The effects of elective cesarean section on lamellar bodies count in amniotic fluid and the expression of ABCA3in term infantsAim:1. To observe the impact of ECS on lamellar bodies count in amniotic fluid on term infants.2. To observe the impact of ECS on the expression of ABCA3of lamellar bodies in term infants.Methods:clinical prospective cohort study1. Lamellar bodies count (LBC) in amniotic fluid (AF) was determined using a conventional hematology analyzer. Then we compared LBC between infants delivered by ECS and those by VD.2. Isolation of LBs in AF was performed by isopycnic equilibrium in a density gradient of sucrose. Then the LB was identified by transmission electron microscope (TEM).3. Flow cytometer analysis of ABCA3expression of lamellar bodies: We added1×106lamellar bodies into each tube, then added20ul PBS into the negative control tubes, or add20ul PBS+lul mouse anti-human ABCA3monoclonal antibody in test tubes, incubated for30min at4℃, washed twice:Added200ul PBS; vortexed, centrifuge at1000rpm for10min and removed supernatant. Added20ul PBS plus lul GAM-FITC in each tube and incubated for30min at4℃in the dark. Washed twice as above and resuspend LB in200ul PBS, followed by the analysis on a flow cytometer (FCM).4. We compared the results of FCM between the infants delivered by ECS and VD.Results:1. LBC in infants with37weeks gestational age, delivered by ECS were lower than those in same gestational age infants delivered by VD (77±23×103/ul, versus 89±35×103/ul, P<0.05). Regardless of delivery mode, LBC in infants with37weeks gestational age were lower than those with more than37weeks gestational age (P<0.05).2. The typical picture of LB was observed by TEM. LB collected from AF was at a size of aboutl-8um (mean5um) and with relatively loose structure, when compared with that in type two alveolar epithelial cell. Some osmiophilic globuli at a size of about0.1-0.5um were also observed inside or outside of LB.3. The average of mean fluorescence intensity (MFI) in positive tube was higher than that of control tube (39.08±10.92, versus10.21±2.14, P<0.05) the percentage of positive cell in positive tube was higher than that in control tube (46.81%±10.75%, versus6.32%±1.42%, P<0.05)4.The mean of MFI of ABCA3in infants delivered by ECS was higher than that in infants delivered by VD (35.2±9.84, versus22.59±4.98, P<0.01), and the percentage of ABCA3positive cell in infants delivered by ESC was higher than that in infants delivered by VD (46.07%±10.31%, versus34.92%±6.69%, P<0.05).Conclusions:1. In term infants, the effects of elective cesarean section on pulmonary surfactant homeostasis were mainly in the infants with37weeks gestational age.2. Regardless of delivery mode, total pulmonary surfactant was still increased after37weeks gestational age in term infants.3. The expression of ABCA3in LB is increased in infants delivered by ESC on the possible mechanisms of compensation.