Lynette Mackay

Cervical ripening : Biochemical, molecular, and clinical considerations

The physiologic and pathologic changes of the uterine cervix during pregnancy leading to cervical ripening are not well understood though are related to the chief pathology and a commonly performed intervention in obstetrics: Preterm birth and labor induction. Normal cervical ripening is thought to be controlled by a variety of hormonal changes occurring during pregnancy leading to softening and dilation. Abnormal premature ripening, usually resulting in preterm labor, is thought to be associated with infection and inflammatory events. Despite many studies of the cervix, we still rely upon relatively crude methods for clinical evaluation of the cervix. In the past several years, we have developed and evaluated a method to measure cervical collagen noninvasively using an instrument called Collascope. Studies in animals and humans conducted in a variety of settings indicate that cervical function can be successfully monitored using the Collascope during pregnancy. We suggest that this technique might be useful to better define management in cases of spontaneous preterm and induced term cervical ripening.

Voltage-clamp studies of gap junctions between uterine muscle cells during term and preterm labor

Gap junctions between myometrial cells increase dramatically during the final stages of pregnancy. To study the functional consequences, we have applied the double-whole-cell voltage-clamp technique to freshly isolated pairs of cells from rat circular and longitudinal myometrium. Junctional conductance was greater between circular muscle-cell pairs from rats delivering either at term (32 +/- 16 nS, mean +/- SD, n = 128) or preterm (26 +/- 17 nS, n = 33) compared with normal preterm (4.7 +/- 7.6 nS, n = 114) and postpartum (6.5 +/- 10 nS, n = 16); cell pairs from the longitudinal layer showed similar differences. The macroscopic gap junction currents decayed slowly from an instantaneous, constant-conductance level to a steady-state level described by quasisymmetrical Boltzmann functions of transjunctional voltage. In half of circular-layer cell pairs, the voltage dependence of myometrial gap junction conductance is more apparent at smaller transjunctional voltages (< 30 mV) than for other tissues expressing mainly connexin-43. This unusual degree of voltage dependence, although slow, operates over time intervals that are physiologically relevant for uterine muscle. Using weakly coupled pairs, we observed two unitary conductance states: 85 pS (85-90% of events) and 25 pS. These measurements of junctional conductance support the hypothesis that heightened electrical coupling between the smooth muscle cells of the uterine wall emerges late in pregnancy, in preparation for the massive, coordinate contractions of labor.

Cervical ripening and insufficiency: from biochemical and molecular studies to in vivo clinical examination.

To understand cervical ripening and especially the pathophysiology of cervical insufficiency, it is important to know the cervical composition: the cervix is dominated by fibrous connective tissue, consisting predominantly of Type I collagen (70%). Despite many studies of the cervix, we still rely upon relatively crude methods for clinical evaluation of the cervix. If the amount of cervical collagen plays a role in cervical insufficiency and in success of or length of induction of labor, then measurements of cervical collagen may provide an objective means of establishing the diagnosis or prognosis. We have established and reported a non-invasive means, called Collascope, to measure collagen cross-linking using light-induced fluorescence (LIF), and which is specifically designed to assess cervical ripening, and functions by measuring the natural fluorescence of non-soluble collagen in the cervix. Studies conducted in animals and humans in a variety of settings indicate that cervical function can be successfully monitored using the Collascope during pregnancy: LIF correlates negatively with gestational age and positively with time-to-delivery interval, and is predictive of delivery within 24h. Additionally LIF is significantly lower in women with cervical insufficiency. We suggest that the Collascope might be useful to better define management in cases of spontaneous preterm or induced term cervical ripening. From our studies and others, it is clear that in forecasting (pre-)term cervical ripening, the capability of the technologies and bioassays that have been generally accepted into clinical practice are limited. Any devices shown to be superior to the clinically accepted tests currently used should be quite useful for clinicians. The Collascope offers an objective measurement of both the function and state of the cervix, by directly measuring collagen cross-linking using LIF.

Effects of progesterone on iNOS, COX-2, and collagen expression in the cervix.

This study examines the relationship between inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the control of cervical ripening and parturition under normal (normal term pregnancy) and abnormal (preterm labor and prolongation of pregnancy) conditions by (a) measuring changes in the collagen both visually and quantitatively, (b) localizing and characterizing iNOS and COX-2 under normal conditions, and (c) characterizing the changes in iNOS and COX-2 under abnormal conditions. Cervices are obtained from estrus and timed pregnant Sprague-Dawley rats (n=4-10 per group). Preterm labor is induced with Onapristone (3 mg/rat; progesterone antagonist) and the prolongation of pregnancy with progesterone (2.5 mg, twice daily). Collagen changes are measured and visualized with the picrosirius polarization method. RT-PCR is used to characterize the mRNA expression (p<0.5), and immunohistochemistry is used to localize the protein expression for iNOS and COX-2. The organization and birefringence of the collagen during pregnancy decreased and is supported by changes in the luminosity (p<0.001). The iNOS and COX-2 enzymes were localized in cervical smooth muscle, vascular smooth muscle, and epithelium. Under normal conditions, iNOS mRNA levels decreased as COX-2 mRNA levels increased demonstrating an inverse correlation (Spearman r = −0.497; p=0.00295). Onapristone stimulated preterm labor, increasing the iNOS and COX-2 mRNA (p<0.05). The increase demonstrated a positive correlation (Spearman r = 0.456; p=0.03). Progesterone prolonged pregnancy, decreasing the iNOS and COX-2 mRNA (p=0.036). In conclusion, there may be an interaction between the nitric oxide and prostaglandin pathways in cervical ripening and parturition.

Gap junction currents in cultured muscle cells from human myometrium

OBJECTIVE The electrophysiologic properties of gap junctions between human myometrial smooth muscle cells were studied. STUDY DESIGN Double whole-cell patch clamp recordings were made on pairs of cells from primary cultures of myometrial cells from women undergoing cesarean section. Macroscopic gap junction currents were measured as the change in current in a cell held at a constant voltage while the other member of a pair was subjected to a test pulse of voltage. The blockade by halothane was examined. RESULTS Mean junctional conductance between pairs of cells was 23 +/- 14 nanosiemens (n = 57). Instantaneous gap junction conductance was constant as a function of transjunctional voltage. For transjunctional voltages of < or = 50 mV, currents were constant during a 5-second test pulse. For larger voltages, however, the currents showed a time-dependent decay. The currents were blocked completely and reversibly with 3.5 mmol/L halothane. Single-channel conductances of 60 picosiemens and 15 picosiemens were observed. CONCLUSION This first study of gap junction currents in human myometrial cells confirms that connexin43 is the major functional constituent. Functional studies of myometrial gap junction channels may suggest new strategies for controlling uterine contractility.