This prospective single-center and randomized clinical trial was approved by the ethical committee of The First Affiliated Hospital of Wenzhou Medical University on 21/09/2021 (Chairperson Pro. Jinglin Xia, NO.KY2021-119) and registered at chictr.org.cn (ChiCTR2100053635) on 26/11/2021. The study was conducted in the First Affiliated Hospital of Wenzhou Medical University between 01/12/2021 and 30/06/2022 according to the criteria of Declaration of Helsinki, and informed consent was obtained from each participant.
Parturients with singleton, full-term pregnancies, ASA physical status I or II, age 20–40 years, height 150–170 cm, weight 50–80 kg, and body mass index (BMI) less than 35 kg/m2 were recruited. Exclusion criteria included the presence of hepatic and renal dysfunction, psychiatric disorders, contraindications for epidural anesthesia, epidural anesthesia failure, the requirement of general anesthesia, prolonged surgical duration (> 1.5 h), and long-term use of analgesics, sedatives, or antidepressants. Those who experienced perioperative hemorrhage, which was defined as a blood loss larger than 500 mL, were also removed.
Randomization and group allocation
Using a simple randomization procedure (1:1 ratio, www.randomization.com), forty-four parturients were enrolled and randomly assigned into either the remifentanil group (Group R) or the control group (Group E). The allocations were sealed in opaque envelopes by the investigator LL and only opened prior to epidural anesthesia by the same attending anesthesiologist (LW) who was not involved with data collection. Epidural anesthesia was planned for the C-section in both two groups. In Group R, an intravenous bolus of 0.15 μg/kg remifentanil was administered for 10 s at skin incision, followed by a continuous infusion of remifentanil at a rate of 0.075 μg/kg/min throughout the procedure, whereas in Group E, the equal volume of normal saline was administered instead. Data collection was performed by JL, XY, ZW, XL, and HZ who were unaware of group allocation.
No premedication was administered, and parturients were required to abstain from food and liquid intake for at least 6 h and 2 h before surgery, respectively. Intravenous access was established and nasal oxygenation at a rate of 3 L/min was administered upon entering the operating room. As a preload prior to anesthesia, 10 mL/kg of Ringer's lactate was administered. The parturient was placed in a left lateral position, and an 18-gauge cannula needle was used to puncture the epidural space, followed by the insertion of an epidural catheter. After administering 3 mL of 2% lidocaine as a test dose via the epidural catheter, an initial bolus of 6 mL 0.5% ropivacaine was delivered, followed by a supplemental dose of 6–9 mL of the same local anesthetic solution to ensure the upper sensory block level of T6. The local anesthetic solution could be administered as needed, up to a safe maximum dose of 200 mg. If the sensory block level failed to reach T6 at 30 min after epidural administration, intravenous analgesics as rescue agents or general anesthesia would be considered and the parturient would be excluded from the study. The patient-controlled epidural analgesia package containing 150 mg ropivacaine and 3 mg morphine in 100 mL normal saline, with a bolus of 2 mL, a background flow of 2 mL/h, and a lockout interval of 15 min, was initiated for postoperative analgesia before transfer to the post-anesthesia care unit (PACU).
Vital signs recording
Mean arterial blood pressure (MAP), heart rate (HR), respiration rate (RR), and oxygen saturation (SpO2) were monitored before anesthesia (T0), at the skin incision (T1), peritoneum incision (T2), neonatal delivery (T3), placental delivery (T4), uterus closure (T5), and abdominal cavity closure (T6).
Visceral pain score
Visceral pain was defined as pain associated with uterine exteriorization and peritoneal traction. The intensity of pain was measured at time points T1-6 using a standard visual analogue scale (VAS) ranging from 0 to 10.
Evaluation of sedation
The sedation level of the parturient was determined using the Ramsay sedation score system, where level 1 indicates anxiety or irritability; level 2 indicates cooperation, quietness, and well orientation; level 3 indicates drowsiness but responsiveness to instructions; level 4 indicates a rapid response to tapping the brow or strong sound stimulation; level 5 indicates delayed response to tapping the brow or strong sound stimulation, and level 6 indicates no response to tapping the brow or strong sound stimulation.
Maternal satisfaction with the anesthetic method
At 24 h postoperatively, maternal satisfaction with the anesthetic method was evaluated using a five-point scale (1 = completely dissatisfied, 2 = dissatisfied, 3 = neutral or undecided, 4 = satisfied, and5 = completely satisfied).
Evaluation of Apgar scores and neonatal asphyxia
The neonatal Apgar scores were recorded at 1 min, 5 min, and 10 min after birth, and the occurrence of neonatal asphyxia was documented. Neonatal asphyxia was diagnosed using the following clinical criteria: neurological abnormalities or neonatal resuscitation required at birth, and/or an Apgar score < 7 at 5 min.
Umbilical arterial pH and the isolation of exosomes
6 mL of blood samples were obtained from the umbilical artery at 5 min after delivery. One portion of each sample was analyzed for pH. UEs were isolated from another portion of umbilical cord sample using ExoQuick exosome precipitation solution (System Biosciences, Palo Alto, CA, USA), a commercially available kit that gently precipitates exosomes ranging in size from 30 and 200 nm . Briefly, umbilical cord serum samples were centrifuged at 3000 g for 15 min, followed by the addition of 250 mL of ExoQuick exosome precipitation solution to 1 mL of the serum supernatants. The mixture was then refrigerated for 30 min and centrifuged at 1500 g for another 30 min. The residual solution was obtained and centrifuged at 1500 g for 5 min to remove the supernatant. The exosome pellet was resuspended in 500 mL of phosphate-buffered saline (PBS) and stored at -80 °C.
Characterization of exosomes using nanoparticle tracking analysis (NTA)
NTA measurements were performed in a flow model using a NanoSight NS300 instrument (Malvern Panalytical, Malvern, United Kingdom) equipped with a 488 nm laser and sCMOS camera module (Malvern Panalytical, Malvern, United Kingdom). Each sample was subjected to NTA at least three times to calculate the mean values. To ensure the accuracy of results, all culture medium samples were identically diluted.
Exosome validation by transmission electron microscopy (TEM)
The morphology of the exosome was identified using TEM. Briefly, a suspension of freshly isolated exosomes was dropped onto a formvar carbon-coated copper electron microscopy grid (Plano, Wetzlar Germany). The grid was then negatively stained for 1 min with 2% uranyl acetate solution, washed with PBS, and dried at room temperature. Finally, images were acquired with an EM 900 transmission electron microscope (Zeiss, Germany) at a voltage of 80-90kv.
Exosomal biomarkers measurement
The total protein content of exosomes was extracted using a homemade cell lysis buffer, followed by protein precipitation using a 5 × protein loading buffer (ABM, Vancouver, Canada). After 5 min of heating in a water bath at 100 °C, the mixture was transferred to a polyvinylidene difluoride membrane (Millipore, Burlington, MA, USA). The PVDF membrane was blocked by incubating it for 1 h at room temperature in milk Tris-buffered saline with Tween20 solution. After that, the membrane was incubated for 15 h with primary antibodies against rabbit anti-mouse CD63 (1:1000; Abcam, USA) and tumor susceptibility gene 101 (TSG101; 1:1000; Abcam, USA). The membranes were then incubated for 1 h at room temperature with HRP-conjugated secondary antibodies (1:1500; Abcam, USA). Finally, the expression of proteins was determined using enhanced chemiluminescence reagents.
Measurement of other outcomes
The adverse events that occurred during surgery and PACU stay were documented. Remifentanil infusion was stopped if the parturient developed respiratory depression, defined as SpO2 < 90% on room air or RR < 8 times/min, the patient was awakened, and ventilation was manually assisted. In addition, hypotension, bradycardia, shivering, cough, nausea, and vomiting were also recorded. (1) Hypotension was defined as a systolic blood pressure < 90 mmHg or a decrease in blood pressure of at least 30% when compared to baseline value, then fluid infusion was accelerated and/or a 0.25 mg bolus of phenylephrine was administered if necessary. (2) If the parturient developed bradycardia, which was defined as a heart rate of less than 60 beats per minute or a drop of more than 30% from baseline, 0.5 mg atropine was then intravenously injected. (3) A four-point scale was used to assess postoperative nausea and vomiting (PONV) (1 = no nausea, 2 = mild nausea, 3 = severe nausea, and 4 = vomiting) . (4) Shivering was also assessed (0 for no shivering, 1 for mild fasciculations of the face or neck, 2 for moderate, visible tremor in more than one muscle group, and 3 for severe, gross muscular activity involving the entire body) . (4) Major side effects of the remifentanil were also documented, including lightheadedness, dyspnea, blurred vision, chest pain, and muscle stiffness and tightness.
The sample size was calculated using the OpenEpi software version 2.3.1 with the number of UEs as the primary outcome. In our preliminary study of 10 cases (n = 5 in each group), the mean number of UEs was 62 × 108 in parturients receiving epidural anesthesia alone, and 99 × 108 in parturients receiving additional remifentanil. We accepted a 5% significance level (two-tailed), an 80% power value, and a 10% dropout rate, resulting in a sample size of n = 22 for each group.
Statistical analysis was performed with SPSS 26.0 software (SPSS Inc, Chicago, IL, USA). The Shapiro–Wilk test was used to examine the normality of the data. Continuous and normally distributed data were reported as mean ± standard deviation (SD), and an independent t-test was performed to compare data between the two groups. Repeated measures analysis of variance was used to compare repeated measured data between different time points within each group, non-normally distributed data were log-normally transformed if necessary before adopting the above statistical methods. Data with non-normal distribution were expressed as median (interquartile range, IQR) and analyzed using the Mann–Whitney U test. Moreover, categorical data were expressed as numbers (%), and Fisher’s exact test was employed to compare these data between the two groups. Statistical significance was defined as P value less than 0.05.