Objective
The purpose of this study was to evaluate cervical changes and delivery at term during pregnancy in rats after various progestin treatments.
Study Design
Pregnant rats were treated by various routes and vehicles with progesterone, 17-alpha-hydroxyprogesterone caproate (17P), R5020, and RU-486. Delivery time was determined and cervical ripening was assessed in vivo by collagen light-induced fluorescence.
Results
The cervix is rigid in the progesterone injection, 17P, and vaginal R5020 groups vs controls. Vaginal progesterone had no effect. RU-486 treatment softened the cervix during preterm delivery. Only subcutaneous injected progesterone, R5020 (subcutaneous and vaginal), and topical progesterone in sesame and fish oil inhibits delivery. Delivery is not changed by subcutaneous injection of 17P, vaginal progesterone, oral progesterone, and topical progesterone in Replens (Crinone; Columbia Labs, Livingston, NJ).
Conclusion
Inhibition of cervical ripening and delivery by progestins depends on many factors that include their properties, the route of administration, and the vehicle. This study suggests reasons that the present treatments for preterm labor are not efficacious.
Preterm birth (<37 completed weeks of gestation) is 1 of the major problems and challenges in obstetrics. The frequency of preterm births is approximately 12-13% in the United States and 5-9% in many other developed countries. Despite all efforts to reduce the number of preterm births, the problem is continuing to escalate. Since 1990 the percentage of children who were delivered preterm has risen >20% and is 36% higher since the early 1980s in the United States. Preterm birth is not only a major determinant of neonatal and infant morbidity (which includes neurodevelopmental handicaps, chronic respiratory problems, intraventricular hemorrhage, infection, retrolental fibroplasia, and necrotizing enterocolitis) but also is the single most important cause of perinatal death in North America, Europe, and particularly in undeveloped countries. Additionally, the neonatal and long-term healthcare costs of preterm infants impose a considerable economic strain both on individual families and on healthcare costs (>$26.2 billion in 2005 in the United States).
Both uterine and cervical functions play important roles in the onset and progression of term and preterm labor and delivery. The cervix undergoes dramatic changes throughout pregnancy and parturition, which is a process that is termed cervical ripening . From a firm, rigid, and closed state that protects the special milieu of the fetus from the environment to a soft and easy-to-open state, that is essential for successful vaginal delivery. The cervix is dominated by fibrous connective tissue that is composed of an extracellular matrix that consists mostly of collagen (70% type I and approximately 30% type III ) with elastin and proteoglycans and a cellular portion that consists of smooth muscles, fibroblasts, epithelium, and blood vessels. Cervical ripening is an active biochemical process that occurs independently of uterine contractions. Studies have shown that cervical ripening is associated with a strong reorganization of the extracellular matrix, especially collagen. Not only does the concentration decrease by 30-70%, but there is also a switch from insoluble to more soluble collagen. Ripening of the cervix is an inflammatory-like reaction with infiltration of leukocytes, an increase of cytokines (interleukin-1 and -8), and an increase in metalloproteinases. This process also seems to be at least partially regulated by steroid hormones (in particular progesterone and estrogen), because antiprogestins successfully induce cervical ripening. Other hormones and mediators that have been shown to be involved in cervical ripening are dihydrotestosterone, prostaglandins, and local mediators such as platelet-activating factor and nitric oxide. Various methods have been used to evaluate cervical ripening and the effects of progestins, including cervical length. We have used light-induced fluorescence (LIF) of the cervix to estimate changes in cervical collagen and effects of treatments. However the biochemical mechanisms that are responsible for the remarkable changes in the cervix remain poorly understood.
Progesterone has long been considered to be a candidate to regulate uterine contractility and cervical function and consequently the onset and progression of labor. Early studies also discussed the potential benefit of 17-α-hydroxyprogesterone caproate (17P), a synthetic caproate ester of the naturally occurring metabolite of progesterone, for the treatment or prevention of preterm labor. The interest in these treatments was renewed by 2 trials that used intramuscular injections of 17P or prophylactic vaginal progesterone for the prevention of preterm labor that confirmed the previous studies by showing a reduction of recurrent preterm birth. In a comment on those trials, Keirse initiated a controversial debate on this topic in which he indicated that “critical analysis of the reports provides no convincing evidence that either one of these treatments is worth pursuing outside the context of controlled research…”. Recently a number of large randomized control trials that used different formulations and routes of administration of progestins have been used to study their effects on reducing preterm delivery. Because of the differences in study populations and drug treatments, it is unclear whether 1 formulation or route of administration is more effective in reducing preterm birth. Pregnant rats are exquisitely sensitive to changes in progesterone with preterm delivery or prolonged gestation when progesterone levels are manipulated or progesterone receptor antagonists are used. The aim of this study was to assess cervical changes throughout pregnancy in rats and the timing of term and preterm delivery after various progestin treatments given by different routes and vehicles in the hope of identifying better treatment regimens.
Materials and Methods
Animals
Nonpregnant and timed-pregnant Sprague-Dawley rats (200-250 g) from Charles-River Laboratories (Wilmington, MA) were delivered to our animal care facilities on day 12 of gestation (day 1 being the day when a sperm plug was observed). The animals were housed separately with free access to food and water and maintained on a constant 12-hour light-dark cycle. Control pregnant rats were spontaneously delivering on day 22 and 23 of gestation. For the measurements with the collascope the animals were anesthetized (intraperitoneal injection) with a combination of xylazine (Gemini; Burns Veterinary Supply Inc, Rockville Center, NY) and ketamine HCl (Ketaset; Fort Dodge Laboratories Inc, Fort Dodge, IA). The animals were allocated randomly to 1 of the groups and killed by carbon dioxide inhalation on day 3, 5, 7, and 10 after delivery or on day 25 of pregnancy in the groups with delayed delivery. All procedures were approved by the Animal Care and Use Committee of the St. Joseph’s Hospital and Medical Center in Phoenix.
Treatments
Before any treatment, LIF measurements were made in control rats throughout pregnancy and the postpartum period to estimate the LIF profile during gestation ( Figure 1 ). Pregnant rats (6 per group) were treated ( Figure 2 ), when not otherwise mentioned ( Figure 3 ), from day 13 of pregnancy until delivery. Single daily treatments were performed at 8 am and twice a day treatments at 8 am and 8 pm . All single injections (4 mg progesterone, 10 mg 17P, 2 mg R5020) were by the subcutaneous route in sesame oil (0.2 mL), which was also used for the controls. Vaginal gels were applied twice a day with a blunt ball-top needle deep into the vagina. The vaginal gel Crinone (Columbia Labs, Livingston, NJ) was used for the progesterone vaginal group (we used equivalent volumes of Crinone for 2-15 mg progesterone per treatment); all data show the results of the highest dose (total daily dose of 30 mg progesterone = one-third of an applicator of 8% Crinone that contains 90 mg progesterone). Another group was treated vaginally with progesterone (30 mg) in 0.5 mL fish oil that was soaked in a cotton plug, inserted each day, and removed before insertion of a fresh one. For the vaginal R5020 group, micronized R5020 (1 mg per treatment) was mixed into 0.18 mL of the vaginal gel Replens. The control rats for the vaginal groups were treated with Replens (0.18 mL per treatment). For oral progesterone treatments (15 mg, twice daily, vehicle sesame oil or water, volume 1 mL), gavage was used. For topical progesterone treatment (15 mg, twice daily, progesterone in 1 mL sesame oil, fish oil, or Replens), the drug was applied on the back of animals that had been shaved on days 13, 17, and 21. In some animals (n = 6) RU-486 (3 mg in 0.2 mL sesame oil) was injected subcutaneously once on day 16 of gestation.
Reagents
Crystalline progesterone (used for oral and subcutaneous progesterone), RU-486, sesame oil, and ethanol were purchased from Sigma Chemical Company (St. Louis, MO); micronized progesterone (used for topical progesterone and vaginal progesterone in cotton plug) was purchased from Spectrum Chemical MFG Corp (Gardena, CA); fish oil (concentrated omega-3 fatty acids) was obtained from General Nutrition Corp (Pittsburgh, PA); 17P was obtained from MP Biomedicals (Solon, OH), and promegestone (R5020) was obtained from Roussel Uclaf SA (Romainville, France). Progesterone, 17P, R5020 and RU-486 were dissolved in ethanol and then mixed with sesame or fish oil. Crinone (micronized progesterone in Replens, a bioadhesive gel, used for vaginal progesterone) and Replens were gifts from Columbia Labs.
Assessment of cervical ripening
The amount of cervical collagen was evaluated in vivo (only in group subcutaneous progesterone, vaginal progesterone, subcutaneous 17P, vaginal R5020, subcutaneous RU-486) by measurement of the autofluorescent properties of cross-linked collagen with a new prototype of an instrument, termed collascope (Reproductive Research Technologies, Houston, TX), as used previously with an earlier prototype. After insertion of a small speculum into the vagina of the anesthetized animal, the optical probe of the collascope was placed on the surface of the exocervix. The probe, which is connected to the main unit of the instrument by a fiber optic cable, delivers not only excitation light (wavelength, 339 nm) onto the cervix but also carries the fluorescent light (mainly caused by pyridinoline cross-links of collagen with a maximum peak at 390 nm) back to the instrument to a charge-coupled device camera to display the full spectrum of fluorescence and analysis of the photons that are emitted by the cervix. In the current study, the exposure time for excitation was 100 msec. The average of 20 measurements of the detected fluorescent intensity (photon count) at 390 nm was used for each animal at any given time. Measurements of cervical LIF were performed on nonpregnant animals once and in pregnant animals every other day starting at day 13 of gestation until delivery and on postpartum day 3 and/or postpartum day 5 ( Figures 2 and 3 ) and for some animals also on postpartum days 7 and 10 ( Figures 1 and 3 ).
Determining the changes in delivery time
Times of delivery (also Figure 4 ) of controls and various treatment groups were determined as hours after 8 am of day 22 of gestation ( Figure 5 ). The expulsion of 1 pup was defined as delivery .
