Objective
To investigate the effect of intracervical hyaluronidase on the biomechanical properties of the cervix and on uterine contractility.
Study Design
Sprague-Dawley rats (n = 33, term day 22) were injected with hyaluronidase (100 IU) or saline solution on day 18 of gestation (n = 8-9/group). On day 21, labor was induced with mifepristone (8 mg/rat). Injection-to-delivery times were recorded. Biomechanical properties of the cervix were assessed using stretch-tension analysis. Myometrial contractility was investigated in response to hyaluronidase (0.2-200 IU/mL), oxytocin (10 −10 M to 10 −5 M), and potassium chloride (60 mM).
Results
Delivery times were shorter in the hyaluronidase group ( P = .03). Cervices of the treated animals showed higher measures of elasticity and plasticity ( P = .02 for both). Myometrial sensitivity to hyaluronidase, oxytocin, or potassium chloride was not affected by the cervical application of hyaluronidase ( P > .05 for all).
Conclusion
Cervical hyaluronidase treatment shortens labor and alters the biomechanical properties of the cervix, independent of the myometrium.
Over the last decade, the rate of labor induction has increased significantly. Early recognition and management of risk factors for maternal and fetal morbidity and mortality, such as a higher incidence of stillbirth beyond 41 weeks of gestation, may at least partially account for this trend. Vaginal delivery cannot be accomplished without ripening of the cervix and stimulation of uterine contractility. The “maturity” of the cervix is commonly assessed by using clinical indicators, such as the Bishop score. It is assumed that if the Bishop score is ≥8 the likelihood of successful vaginal delivery after labor induction will be similar to that of a spontaneous labor. However, the need for induction of labor in the face of an unripe cervix is a common clinical dilemma, either at term or preterm. Currently used methods of cervical maturation include mechanical dilators, synthetic prostaglandin E 1 and E 2 preparations, and intravenous oxytocin. Identification of novel pharmacologic agents that allow for a safe process of cervical ripening and labor induction is critically needed.
The biology of the cervix undergoes major molecular, enzymatic, and biomechanical transformations that differ from those of the myometrium. Stromal cells, smooth muscle, blood vessels, and an extracellular matrix consisting of collagen, elastin, and glycosaminoglycans (GAGs) are major structural components of the cervix. Initial studies on the molecular mechanisms involved in the process of cervical ripening focused primarily on changes in the collagen component of the uterine cervix. As our understanding of the process of physiologic cervical ripening improved, we have gained a greater appreciation for the noncollagen component of the extracellular matrix, which is composed mainly of GAGs. There is consensus that the physical properties of the cervix depend on the interplay between collagen and GAGs. Hyaluronic acid (HA), one of the chief components of the extracellular matrix, is a GAG distributed widely throughout connective tissues, including those of the uterine cervix. HA has a high affinity for water molecules and therefore may control local tissue hydration, which is an essential component of the process of cervical ripening.
Hyaluronidase (HAase) is an intrinsic enzyme that catalyzes the hydrolysis of HA, effectively creating low-molecular-weight HA. This catalyzing action lowers the viscosity of HA, thus increasing tissue permeability to water. Recent studies suggest that this enzyme may be useful as a cervical ripening agent. Still, the impact of HAase on the viscoelastic properties of the cervix and on the myometrial sensitivity to agonists and antagonists remains unknown.
Our objective was to test the hypothesis that intracervical application of HAase shortens the duration of the labor induction process by modifying the biomechanical properties of the cervix independent of myometrial contractility. We tested our hypothesis in a rat model of labor induced using the antiprogesterone mifepristone.
Materials and Methods
Animals
Adult, timed pregnant nulliparous Sprague-Dawley rats (n = 33, term day [d] 22, d0 = day sperm plug observed) weighing 250-350 g were acquired from Charles-River (Wilmington, MA) on d14 of pregnancy. They were housed separately in temperature- and humidity-controlled quarters with constant light:dark cycles of 12 hours:12 hours and provided with food and water ad libitum. All the procedures were approved by the Institutional Animal Care and Use Committee at The University of Texas Medical Branch.
Labor induction
On d18 of gestation, 16 rats were randomly allocated into 2 groups (n = 8/group). Animals were anesthetized with ketamine (22.5 mg; Phoenix Scientific, Inc, St. Joseph, MO) and xylazine (1.5 mg; Phoenix Scientific, Inc). After pain control, the cervix was visualized with the aid of a cone-shaped plastic guide. Under sterile conditions, the HAase group animals received an injection of 100 IU of HAase given as 2 separate injections into the anterior and posterior lips of the cervix. We used lyophilized HAase enzyme (Vitrase; ISTA Pharmaceuticals, Albuquerque, NM) that was resuspended with normal saline (total volume 1.0 mL). The dose of treatment was chosen based on a prior study published by de Souza et al. Under similar experimental conditions, the control (CRL) animals received an intracervical injection of 1.0 mL normal saline solution, given as 2 separate injections into the anterior and posterior lips of the cervix. All the injections were placed at a depth of approximately 2-3 mm into the cervical stroma. To avoid spillage, the 18-gauge needle was left in place for 30 more seconds after injection of HAase or saline solution.
Seventy-two hours (d21) after treatment, labor was induced in all animals using a subcutaneous injection of the antiprogesterone agent mifepristone (8 mg; Sigma-Aldrich Chemical, St. Louis, MO) as previously described. After mifepristone administration, the HAase and the CRL animals were observed until completion of the labor process. Labor was considered complete when the animal delivered its last pup.
Testing of the biomechanical properties of the cervix and myometrium
A separate group of rats (n = 17) were randomly distributed into 2 groups (n = 8-9/group), and their cervices were injected on d18 with HAase or saline solution in the same fashion as described previously. On d21, all rats were euthanized using CO 2 inhalation. Immediately after death, the cervix and the uterus were surgically removed. The cervix was defined as the less vascular tissue with parallel lumina between the uterine horns and the vagina. The uterine horns were identified and, for consistency, the site immediately distal to the third fetus on the right horn was isolated for the myometrial experiments. The tensile properties of the isolated cervix and myometrium were evaluated based on a previously described protocol. Briefly, each cervical and myometrial ring sample was anchored in a tissue bath containing 10 nM of N-2-hydroxyethylypipera-zine-N-2-ethane sulfonic acid/phosphate-buffered saline solution (HEPES, pH 7.4) by a 2-0 silk thread passing through each of the 2 lumens of the cervical specimens or through the single lumen of the myometrial specimen. A Shimadzu EZ-test instrument (Shimadzu North America, Columbia, MD) was used to stretch the tissue at a rate of 0.42 mm/min (sampling rate: 20 Hz; duration of stretching: 7 seconds; duration of equilibration: 59 seconds). This stretching protocol was designed to mimic the attributes of labor and is a modified version of the original protocol designed by Downing and Sherwoood. Biomechanical parameters such as slope (measure of viscoelasticity), yield point (YP; moment when tissue changes from elastic to plastic), break point (BP; measure of tissue strength), and displacement at YP were recorded and analyzed. Plateau (measurement of the plastic phase) was defined as the difference between the BP and the displacement at YP. Once initiation of stretching for each cervical or myometrial sample was begun, it was carried out past the YP until the BP was reached (when the tissue visibly breaks). An average of the upper and lower slope was calculated to characterize the sample’s resistance to stretch. All data were normalized to the wet weight of the specimen.
Myometrial contractility studies
Using the same animals tested for the cervical biomechanical experiments described above, 2 uterine rings of about 5 mm width were consistently obtained from each animal at a site just proximal to the segment removed for the biomechanical experiments. Rings were mounted vertically in 10 mL organ chambers containing Krebs solution using stainless steel hooks. One end of the strip was attached to a fixed support at the bottom of the chamber, whereas the other end was connected to an isometric force transducer. The temperature in the organ bath was maintained at 37°C, and the solution was continuously bubbled with gas (5% CO 2 in air, pH ~ 7.4). Isometric tension was measured using Harvard isometric force transducers (Harvard Apparatus, South Natik, MA) connected to a computer. The data were acquired and analyzed using Windaq data acquisition software (Dataq Instruments, Inc, Akron, OH). The strips were equilibrated at the passive tension of 2 g for 1-2 hours until spontaneous regular and rhythmic contractions were observed for at least 30 minutes (contraction stabilization). The bath solution was changed every 30 minutes. Concentration response curves to cumulative doses of oxytocin (10 −10 M to 10 −5 M) and HAase (0.2-200 IU/mL) were determined. Comparative analyses were performed between HAase-treated and CRL animals. At the end of each experiment, KCl (60 mM) was added in each muscle bath chamber to confirm tissue viability. Myometrial strip activity was recorded for 30 minutes.
Data analysis
Normality of the data distribution was tested, and the data are presented as mean ± standard deviation (SD). Statistical analysis was performed using Student t test or 2-way analysis of variance (ANOVA) as appropriate. SigmaSTAT 2.03 statistical software (Jandel Corp, San Rafael, CA) and GraphPad Prism version 4.00 for Windows (GraphPad Software, La Jolla, CA) were used. A P value of < .05 was considered statistically significant.
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