Objective
The objective of the study was to determine the effects of electrical stimulation (ES) on cervical ripening in pregnant and nonpregnant rats.
Study Design
Timed pregnant and nonpregnant Sprague-Dawley rats (n = 6–7/group) were used. Cervical ES for pregnant rats was performed in vivo on day 15 of gestation by inserting an electrical probe into the vagina in contact with the cervix. Parameters of ES varied from 0.1 to 0.2 mA, 10 pulses per second, 20 milliseconds pulse duration, and repeating pulses for 15, 30, 60, and 120 minutes for pregnant ES groups and similar times for sham control groups with electrode but without ES. Nonpregnant ES groups were stimulated with only 0.2 mA for 30 minutes. Cervical collagen was measured in controls and following ES at various times using light-induced fluorescence (LIF) of collagen. Photographs were taken following ES, and some rats were killed, the cervices were isolated, and cervical extensibility was estimated.
Results
LIF values of pregnant rats are significantly lower ( P < .001) and extensibility greater ( P < .05) in the ES treatment groups compared with the control groups on days 16 and 17 of pregnancy. Similarly LIF is lower ( P < .05) and extensibility values greater ( P < .05) in nonpregnant rats treated with ES. No adverse effects, including altered delivery time, pup weights, or damage to cervix, were produced by low current levels of ES needed to soften the cervix.
Conclusion
The following conclusions were reached: (1) application of ES rapidly produces softening and ripening of the cervix in pregnant and nonpregnant rats; (2) ES treatment does not produce early delivery; (3) the exact mechanism for ES ripening is not yet known; and (4) ES might be used clinically to ripen the cervix when needed.
Softening, effacement, and dilation are critical and sequential steps included in the ripening process of the cervix to convert it from a rigid to an extensible state and are necessary for initiation of parturition and normal delivery. Cervical softening, a chronic event, usually occurs progressively during pregnancy and begins in early pregnancy, whereas effacement and dilation of the cervix are more acute changes that occur near term.
The steps from softening to dilation provide a pliable and accessible pathway for delivery of the fetus. Generally, ripening that occurs too early can often lead to preterm birth and delay in ripening progresses to postterm pregnancy with complications including substantial increase in perinatal mortality and morbidity, such as meconium-stained amniotic fluid with aspiration, which sometimes proceeds to cesarean delivery. In addition, a lack of sufficient cervical ripening can advance to gestational age increase or postterm pregnancy, which is associated with unexpected stillbirth. Therefore, it is often desirable to artificially promote cervical ripening.
The cervix of all mammalian species, including humans, is composed of connective tissue and a small proportion of smooth muscle that is continuous from the uterus through the vaginal wall. Changes in connective tissue and water content of the cervix are responsible for the softening process during pregnancy. Concentrations of collagen, glycosaminoglycans, and hyaluronic acid decrease during pregnancy to result in a more pliable organ capable of effacement and dilation. In humans, the concentration of collagen has been estimated at approximately 70% at 10 weeks of gestation and at term approximately 30% compared with that of nonpregnant patients. Physiologically the softening process and ripening of the cervix are thought to be followed by uterine contractions of labor. Therefore, it is desirable to have a soft cervix before the uterus begins to forcefully contract to accomplish the delivery process.
In animals the process of cervical ripening also proceeds as in humans but on a shorter time frame, depending on the length of gestation. Many studies of rats, with a gestation period of 22 days, have shown that the cervix of pregnant animals in early gestation is softer than that of nonpregnant animals and slowly becomes more extensible until term. Many approaches have been used to promote or delay cervical ripening in various species.
Clinically, various mechanical or pharmacological methods, including the use of mechanical devices such as a Foley catheter, hygroscopic and osmotic dilators, and pharmacological agents such as misoprostol, dinoprostone, oxytocin, and nitric oxide donors are frequently applied to ripen the rigid cervix and induce labor in humans during pregnancy when it is indicated. However, some of them have undesirable side effects to the mother and/or fetus.
Different methods of softening the rigid cervix are also used in nonpregnant patients when entry into the uterine cavity is clinically relevant. Ideally a method that ripens the cervix should soften the cervix without causing unnecessary pain and have minimal side effects on the mother or fetus. It is generally agreed that a better method for ripening the cervix would lead to a reduction in the number of cesarean deliveries.
Electrical stimulation (ES) is well known to elicit responses in a variety of excitable tissues, such as nerve, skeletal, and cardiac muscle. ES has also been studied for action on various types of smooth muscle, including cervical, airway, bladder, myometrial, gastrointestinal, and other smooth muscle tissues. In addition, ES (transcutaneous electrical nerve stimulation) has been used in obstetrics to relieve pain during labor as well for a diversity of other treatments including bone and tissue repair and collagen synthesis. Therefore, we suspect that ES may be safely used for cervical ripening by physically or chemically changing the composition of the cervix.
The aims of this study were as follows: (1) to determine whether ES would ripen the cervix in pregnant and nonpregnant rats; (2) to confirm that ES softens the cervix by measuring changes in cervical extensibility; and (3) to examine the effects of ES on the timing of parturition and damage to the cervix and determine any fetal effects.
Materials and Methods
Animals
Timed-pregnant Sprague-Dawley rats (240–280 g) from Charles River Laboratories (Wilmington, MA) were delivered to our animal care facilities on day 13 of gestation (day 1 being the day when a sperm plug was observed). The rats were randomly divided by a technician blinded to the study design into groups (n = 6–7 per group). The animals were housed separately, with free access to food and water and maintained on a constant 12 hour light-dark cycle.
During ES the rats were lightly anesthetized with intraperitoneal injections of a mixture ketamine (Ketalar; Parke-Davis, Morris Plains, NJ) and xylazine (Gemini; Burns Veterinary Supply, Inc, Rockville Centre, NY) to keep the rats quiet and immobilized during treatments. Similarly, a limited number of non-pregnant rats (n = 6 per group) were also used, at indeterminate stages of the estrus cycle, to estimate the effects of ES. The rats were killed by CO 2 inhalation after delivery or treatment.
All procedures were approved by the Animal Care and Use Committee of the St. Joseph’s Hospital and Medical Center (Phoenix, AZ). In this study, various groups of pregnant and nonpregnant rats were studied (total rats = 73 pregnant and 12 nonpregnant; Results , Figures 1-7 and Table ).
| Group | Time, min | Control | ES | Stimulation, mA | P value |
|---|---|---|---|---|---|
| 15 | 60.50 ± 1.00 | 58.23 ± 0.55 | 0.1 | .09 | |
| Pregnant | 56.53 ± 1.00 | 0.2 | .006 | ||
| 30 | 62.45 ± 2.00 | 52.69 ± 1.00 | 0.1 | < .001 | |
| 54.91 ± 1.40 | 0.2 | .003 | |||
| 60 | 60.84 ± 3.73 | 51.54 ± 1.22 | 0.1 | .02 | |
| 45.56 ± 2.01 | 0.2 | < .001 | |||
| Nonpregnant | 30 | 92.84 ± 1.97 | 81.86 ± 2.07 | 0.2 | .009 |
Electrical Stimulation
ES was achieved with the use of a Grass S-88 stimulator connected to a constant current unit (Grass Technologies, West Warwick, RI; now Natus Neurology Inc, Middleton, WI). Cervical ES was performed in vivo in pregnant rats on day 15 of gestation by inserting an electrical probe (Grass Technologies bipolar stimulation electrode, model F-BSE1 with 2 mm diameter polished gold-plated tips set at 2 mm apart) into the vagina (dilation of vagina was achieved with an infant nasal speculum) in contact with the cervix.
For the procedure mentioned in previous text, the rats were placed on their backs and taped to a tabletop, and the probe was held in place by clamping the external portion of it to a rigid support stand. To avoid any damage to the cervix, according to the results of preliminary tests, current was applied at 0.1 mA and 0.2 mA with a frequency of 10 pulses per second and pulse duration of 20 milliseconds and repeating pulses for 15, 30, and 60 minutes for pregnant ES groups and the same time frames for control groups (sham controls) with electrode but without ES. Based on studies with pregnant rats, nonpregnant ES groups were stimulated with only 0.2 mA of current for 30 minutes and nonpregnant sham controls used with instrumentation but without ES.
In this study, we used a stimulation frequency of 10 pulses per second (10 Hz) with each stimulus pulse duration at 10 to 20 milliseconds. These stimulus pulse duration parameters were chosen because the stimulus duration to activate excitable tissues is dependent on stimulus strength vs duration of pulse width curves to define the chronaxie of the tissue. Chronaxie is the stimulus pulse duration at which the threshold intensity is twice the lowest intensity with indefinite pulse duration, which just activate muscles or nerves (rheobase).
Chronaxie is an important consideration in pacing the stimulation of any tissue because chronaxie defines the stimulation pulse duration parameter required to activate an excitable tissue. Chronaxie values are dependent on stimulation to open voltage-dependent channels in excitable cells and thereby activate them. Chronaxie values vary in excitable tissues in the following order: nerves less than skeletal muscle less than cardiac muscle less than smooth muscle with nerves requiring a shorter duration pulse (ca. 40–200 microseconds) for excitation and smooth muscle longer durations.
Measurements of cervical light–induced fluorescence and extensibility
In pregnant rats, cervical collagen was measured in controls and following ES using light-induced fluorescence (LIF) of collagen with a collascope as used previously. Similarly, LIF was used in nonpregnant rats. To confirm that ES softens the cervix as determined by LIF, resistance to stretch (extensibility) was estimated in control and ES-treated rats. On the second day following ES stimulation, the rats were killed, the cervices were isolated. and resistance to stretch were estimated by measuring extensibility by increasing the tension on cervices for 5 seconds at 4 minute intervals. The slopes of the regression line through the peaks of the force/length curves were estimated, indicating extensibility of the tissue.
Effects of ES on timing of birth, damage to cervix, fetal weights, and adverse events
Some rats treated with ES or their controls were followed daily until delivery and fetal weights and viability recorded. To assess any damage caused to the cervix by ES, photographs of the cervix of each rat were taken before and following ES with a small endoscopic camera. Rats were observed daily for instances of vaginal bleeding and early fetal loss. Photographs were also taken of all litters following birth.
Measurement of uterine electrical and contractile activity
Some pregnant rats (n = 5) were fitted an intrauterine pressure catheters (SPR-320 rat pressure catheter, size 2F; Millar Inc, Houston, TX) placed surgically within the uterine cavity in the middle of 1 uterine horn between the inner uterine wall and fetal membranes. Signals from the pressure catheter were converted to pressure (millimeters of mercury) using procedures recommended by Millar Inc with a calibrated pressure gauge.
Electromyographic (EMG) activity was obtained with multistranded bipolar stainless steel wire (0.009 inch diameter, catalog no. 793500; A-M Systems, Sequim, WA) attached directly to the uterine surface near the pressure catheter to measure uterine electrical activity (band pass filtered at 0.3–2.0 Hz and sampled at 100 Hz) during ES. Both pressure and uterine EMG were recorded simultaneously with a PowerLab data acquisition system with LabChart software (AD Instruments, Bella Vista, NSW, Australia). The intrauterine pressure and EMG activity were measured before, during, and following ES and similarly in the control rats without ES.
Statistical analysis
Results are expressed as means ± SEM. Data analysis between multiple groups were done using a 1-way analysis of variance followed by a Student-Newman-Keuls test. Data were analyzed for statistical significance using a Sigma Stat statistical software program (version 3.01A; Richmond, CA). Statistical significance was defined as P < .05.
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