Objective
The purpose of this study was to determine whether optical methods can estimate cervix function during pregnancy and whether progestins modify this process.
Study Design
Photos of the external cervix of timed-pregnant rats were taken every other day from day 13 until postpartum day 5 after daily treatments with vehicle (controls) or progestin treatments (progesterone, subcutaneously or vaginally; 17-alpha-hydroxyprogesterone caproate [17P] and RU-486 subcutaneously, once on day 16). The surface area of the cervix was estimated from photos.
Results
The surface area of cervix increases throughout pregnancy and reverses after delivery in controls. In the progesterone subcutaneously or 17P subcutaneously groups, increases in surface area are lower (17P group until day 19 only; P < .05). Vaginal progesterone does not prevent surface area increases. Only the progesterone subcutaneously blocked delivery. RU-486 increases the surface area of the cervix ( P < .05) during preterm delivery.
Conclusion
An optical method is useful for quantitative assessment of the cervix and evaluation of agents that modify cervical function.
Preterm birth is a severe pregnancy complication that occurs in approximately 10% of all pregnancies in the developed countries and even more often in developing countries. Despite all efforts, at this date reliable tools that predict and diagnose preterm birth and successful treatment regiments for a better outcome of the pregnant woman and the baby are still missing. The development of effective therapies to prevent or reduce the occurrence of this difficult medical condition depends on the understanding of the circumstances that initiate labor.
After staying rigid and closed throughout most of pregnancy, to protect and secure the special environment created inside the uterus, the cervix switches to a soft and easy-to-open state that is essential for successful vaginal delivery. Many biochemical and functional changes occur in the cervical connective tissue during gestation, which are summarized in the term cervical ripening . It is a chronic process that starts in the first trimester of pregnancy and progressively proceeds until term. It is usually described as a 3-step preparation process that must occur in sequence, and each step seems to be irreversible: softening, effacement, and dilation of the cervix. This sequence is associated with a dramatic reorganization of the extracellular matrix, which consists of elastin, proteoglycans, and especially collagen, that decreases by 30-70% and is accountable by a change from insoluble to more soluble collagen. Cervical ripening is an active biochemical process with similarities to an inflammatory-like reaction (infiltration of leukocytes, increase of cytokines and metalloproteinases) and occurs independent of uterine contractions. This process also appears 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.
The consistent and precise identification of the changes that occur in the cervix is one of the challenges that obstetricians face today. Various methods have been used to identify this condition. Physical examination is one of the oldest techniques, and a number of scoring systems to characterize the cervix (eg, the Bishop Score) have been developed. Other techniques to assess cervical changes are the measurement of the cervical length by transvaginal ultrasound scans and biochemical markers (such as fetal fibronectin, which is a glycoprotein that is found at the chorionic-decidual interface, or insulin-like growth factor binding protein-1, which is a protein that is synthesized by the maternal decidua). Our group has used light-induced fluorescence (LIF) of the cervix to estimate changes in cervical collagen and effects of treatments. These studies show that parenteral or topical progesterones are equally effective in inhibiting delivery in rats but that these treatments only partially prevent cervical ripening.
Recent studies have investigated the use of progestins as treatment for preterm delivery. Early studies also discussed the potential benefit of 17-α hydroxyprogesterone caproate (17P), which is a synthetic caproate ester of the naturally occurring metabolite of progesterone, for the treatment or prevention of preterm labor. Several recent randomized controlled trials have studied the effects of progestins on cervical length changes that were assessed by transvaginal ultrasound scanning. Other possible treatments for woman who are at risk of cervical insufficiency include bed rest, cervical cerclage, and antibiotics. None of these reports are evidence-based, and there is a controversy in the findings. Uterine contractions can be suppressed by tocolytic drugs, but only for a very limited time and all compounds have considerable side-effects.
Pregnant rats, which are a well-known model to study pregnancy in animals, are exquisitely sensitive to changes in progesterone with preterm delivery or prolonged gestation when progesterone levels are manipulated or when progesterone receptor antagonists are used. Our hypothesis is that exogenous progestins inhibit cervical ripening and prevent term delivery, that there are differences in abilities of the progestins, and that the consideration of routes of administration is important.
The objective of this study was to determine whether optical methods can be used to estimate changes of the properties of cervical tissue during pregnancy and whether progestins that are given by various routes can alter these properties. We also used LIF to assess cervical changes and compared them with an optical surface analysis of changes in the area of the cervix during pregnancy and after delivery.
Materials and Methods
Animals
Timed-pregnant Sprague-Dawley rats (200-250 g) from Charles-River Laboratories (Wilmington, MA) were transferred 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 spontaneously delivered on days 22 and 23 of gestation. For the measurements with the endoscopic camera and 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 postpartum day 5 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, AZ.
Treatments
Pregnant rats (n = 6/group) were treated (when not otherwise mentioned) from day 13 of pregnancy until delivery. Single daily treatments were performed at 8 am , and twice daily treatments were performed at 8 am and 8 pm . All daily injections (4 mg progesterone and 10 mg 17P) were by the subcutaneous route in sesame oil (0.2 mL), which was also used for the controls of the injection groups. Vaginal gels were applied twice a day with a blunt ball-top needle deep into the vagina. Crinone was used for the progesterone vaginal group (we used equivalent volumes of Crinone for 2-15 mg progesterone/treatment); all data presented show the results of the highest dose (total daily dose of 30 mg progesterone = one-third of an applicator of 8% Crinone that contained 90 mg progesterone). The control rats for the vaginal groups were treated with Replens (0.18 mL/treatment). RU-486 (3 mg in 0.2 mL sesame oil) was injected subcutaneously once on day 16 of gestation.
Reagents
Crystalline progesterone (used for subcutaneous progesterone), RU-486, sesame oil, and ethanol were purchased from Sigma Chemical Company (St. Louis, MO). 17P was obtained from MP Biomedicals (Solon, OH). Progesterone, 17P, and RU-486 were dissolved in ethanol and then mixed with sesame oil. Crinone (micronized progesterone in Replens, which is a bioadhesive gel, was used for vaginal progesterone) and Replens were gifts from Columbia Laboratories (Livingston, NJ).
Assessment of cervical changes
Measurements with the endoscopic camera and the collascope were performed on every other day starting at day 13 until day 21 of gestation and on postpartum day 3 and/or postpartum day 5 and for some animals also on postpartum days 4, 8, and 10.
Optical evaluation with an endoscopic camera
A small pediatric speculum was inserted into the vagina of the anesthetized animal and always opened to a certain level (distance between the top and the bottom part of the end of the speculum was set to standard width of 9 mm, which was used for calibration of the measurements of the surface area). An endoscopic camera was placed in front of the cervix at approximately 10 mm, and photos were taken of the cervix and ends of the speculum ( Figure 1 ). The surface area (in square millimeters) of the cervix was calculated from digitized photographs by morphometric methods with the use of ImageJ software (version 1.43; National Institutes of Health, Bethesda, MD).
Evaluation with the collascope with LIF
The amount of cervical collagen was evaluated 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 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 excitation light (wavelength, 339 nm) onto the cervix and 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. 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.
Determining the changes in delivery time
Pregnant animals were checked for delivery 3 times per day ( 8 am, 12 am, 8 pm ). The expulsion of 1 pup was considered to be the start of delivery.
Analysis
The surface area of the cervix and the LIF of control animals ( Figure 2 ) that were obtained at different times of gestation were compared by 1-way analysis of variance and multiple pairwise comparison procedures (Dunn’s Method for cervical surface area and Holm-Sidak for cervical LIF). The Student t test was used to compare the surface area of a treatment group to its specific control group at any time in gestation and after delivery ( Figure 3 ) and to determine the differences in delivery times ( Figure 4 ). A 2-tailed probability value of < .05 was considered statistically significant.
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