Direct electrical stimulation softens the cervix in pregnant and nonpregnant rats.

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.

Nicotine, an α7 nAChR agonist, reduces lipopolysaccharide-induced inflammatory responses and protects fetuses in pregnant rats.

OBJECTIVE: The objective of the study was to examine the effects of nicotine, an α7 nicotinic acetylcholine receptor agonist, on lipopolysaccharide (LPS)-induced inflammatory responses in rats during pregnancy. STUDY DESIGN: Pregnant Sprague Dawley rats were randomly divided into groups (n = 6 rats/group): group 1 rats each received a single intraperitoneal injection of LPS (25 µg/kg) on gestation day 16; group 2 rats were first pretreated with nicotine (1 mg/kg per day, subcutaneously) on gestation days 14 and 15 and then were treated with single injections of LPS on gestational day 16; group 3 rats were treated with the vehicle (saline) used for groups 2 and 3 (controls). Maternal blood was collected at 6 hours and 24 hours after LPS and vehicle treatments and assayed for tumor necrosis factor (TNF)-α, interleukin-6 (IL-6), and interleukin-10 (IL-10). In addition, the number of live pups and pup weights were obtained at the time of delivery. RESULTS: LPS treatment significantly (P < .001) elevates maternal blood levels of TNF-α and IL-6 but not IL-10 (P > .05). Nicotine treatment significantly reduces LPS-induced TNF-α and IL-6 concentrations (P < .001) but does not change (P > .05) IL-10 levels. The number of live pups in the LPS group are significantly lower (P < .001) than the vehicle treated controls, and nicotine treatment significantly (P < .011) reverses this change. Similarly, fetal weights are lower following LPS (P < .016) and higher (P < .024) in the group treated with nicotine plus LPS. CONCLUSION: Nicotine reduces the LPS-induced inflammatory responses and rescues the fetus in rats during pregnancy. Thus, nicotine exerts dramatic antiinflammatory effects. These observations have important implications for control of inflammatory responses during pregnancy.

Nicotine treatment prolongs gestation and inhibits cervical ripening in pregnant rats.

OBJECTIVE: The aims of this study were to examine the effects of nicotine treatment on the length of gestation, on fetal outcome, on cervical ripening, and on uterine contractility during pregnancy in rats. STUDY DESIGN: Pregnant rats were treated with various concentrations of nicotine (0.25, 0.5, 1, 2 mg/kg/d, subcutaneously). Delivery times and fetal weights were obtained. Cervical collagen cross-links were assessed in vivo by collagen light-induced fluorescence (LIF), and cervical resistance to stretch was measured by in vitro extensibility tests. RESULTS: Delivery time is significantly (P = .002) prolonged after high-dose nicotine treatments. There are no significant changes in pup weights and placenta weights after nicotine treatments. Cervical collagen LIF and extensibility progressively decrease throughout pregnancy in control rats. Nicotine-treated rats showed significant (P < .001) cervical resistance to stretch and higher LIF compared with the control rats. Nicotine treatment in vitro had little effect on uterine contractility. CONCLUSION: Nicotine exposure during pregnancy prolongs gestation and inhibits cervical ripening, possibly by suppression of a cholinergic antiinflammatory response.

Inhibition of uterine contractility with various tocolytics with and without progesterone: in vitro studies.

OBJECTIVE: Various tocolytics are used to suppress uterine contractility in patients in preterm labor. Progesterone (P4) is used in patients at high risk for preterm delivery. In this study, we evaluated the effects of various tocolytics with and without P4 to examine effects on uterine contractility. STUDY DESIGN: Uterine tissues (n = 280) from women undergoing cesarean at term were exposed in vitro to various agents (vehicle, magnesium sulfate [MgSO(4)], nifedipine, indomethacin, or pinacidil-all with and without P4). Contractility was measured before and after addition of the various agents. RESULTS: P4 alone at 10(-5) mol/L concentration has little effect to inhibit contractility (P ≥ .05). MgSO(4) (2-8 × 10(-3) mol/L) inhibits uterine contractility (P < .05) but there is no change when combined with P4 (P > .05). Nifedipine (10(-8) mol/L) and indomethacin (10(-5) mol/L) inhibit contractions alone (P < .05) and to a greater extent when combined with P4 (P < .05). P4 significantly (P < .05) reduced the effects of pinacidil (10(-6.5) mol/L). CONCLUSION: Combinations of P4 with nifedipine or indomethacin, but not MgSO(4), might be used to effectively suppress preterm labor.

Progestin treatment for the prevention of preterm birth.

Progestin supplementation appears to be a promising approach to both preventing initiation of preterm labor and treating it once it is already established, given the role of progesterone in maintaining pregnancy, as well as support from basic and clinical research. Progesterone and 17α-hydroxyprogesterone acetate slow the process of cervical ripening, and this is the rationale for prophylactic long-term progestin supplementation mostly studied so far. However, progesterone (but not 17α-hydroxyprogesterone acetate) also inhibits myometrial activity even after the cervix has already ripened. Moreover, these effects depend greatly on the vehicle used and the route of administration. Understanding different mechanisms of action, as well as the importance of progestin formulation, vehicle and route of administration, is the key to finding the optimal progestin treatment for prevention of preterm birth.

A novel optical method to assess cervical changes during pregnancy and use to evaluate the effects of progestins on term and preterm labor.

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.

Additive inhibitory effects of progesterone and sodium nitroprusside on uterine contractility during pregnancy.

Progesterone (P4) and nitric oxide (NO) suppress uterine contractility (CTX). This study compares the effects of P4 to sodium nitroprusside (SNP, an NO donor) and their combination on human CTX of term/preterm and labor/nonlabor tissues. Uterine tissues (n = 128) from women (n = 28) undergoing Cesarean were suspended in organ baths. Tissues (n ≥ 6/group) were treated with vehicle, P4, SNP, or combinations. A subset of tissues (n ≥ 2/group) from term/preterm ± labor and nonpregnant patients was analyzed with P4 alone. Analysis of variance (ANOVA) was used for statistical differences (P < .05). The combination of P4 with SNP significantly suppresses CTX (% inhibition of -127.1 ± 14.5) to the levels lower than with either P4 (-20.1 ± 8.6) or SNP alone (-72.0 ± 11.2). Suppression of P4 is similar in term, preterm, and nonpregnant tissues, with increased sensitivity in laboring tissues. This indicates that P4 or SNP alone may be used for preterm labor and their combination may be more successful.

Use of uterine electromyography to diagnose term and preterm labor.

Current methodologies to assess the process of labor, such as tocodynamometry or intrauterine pressure catheters, fetal fibronectin, cervical length measurement and digital cervical examination, have several major drawbacks. They only measure the onset of labor indirectly and do not detect cellular changes characteristic of true labor. Consequently, their predictive values for term or preterm delivery are poor. Uterine contractions are a result of the electrical activity within the myometrium. Measurement of uterine electromyography (EMG) has been shown to detect contractions as accurately as the currently used methods. In addition, changes in cell excitability and coupling required for effective contractions that lead to delivery are reflected in changes of several EMG parameters. Use of uterine EMG can help to identify patients in true labor better than any other method presently employed in the clinic.

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.

The effect of bilateral pelvic neurectomy on cervical ripening in pregnant rats.

AIMS: To determine the effect of bilateral pelvic neurectomy (BPN) on cervical ripening in pregnant rats by measuring cervical extensibility and changes in collagen cross-linkages. METHODS: Timed-pregnant rats were randomly laparotomized on days 9 or 10 of gestation and the pelvic nerves were exposed and either bilaterally transected, or left intact in sham control animals. The rats were sacrificed on day 18 and the uterine cervices obtained. Cervical ripening was assessed by cervical resistance-to-stretch, light-induced autofluorescence (LIF) of cross-linked collagen, and collagen changes analyzed by picrosirius polarization microscopy. RESULTS: Measurements of extensibility and collagen cross-linkages indicated that after BPN the cervix was significantly more ripened than the cervix from sham control animals. CONCLUSION: BPN stimulates cervical ripening instead of inhibiting this event as previously proposed. Further studies in this area could be critical for developing treatments for dystocia, preterm labor, and cervical insufficiency.

Treatment with an inhibitor of catechol-O-methyltransferase activity reduces preterm birth and impedes cervical resistance to stretch in pregnant rats.

Catechol-O-methyltransferase (COMT) enzyme catalyzes the methylation of the 2- or 4-hydroxyestrogens to 2- or 4-methoxyestrogens. Both the hydroxyestrogens and methoxyestrogens have been shown to block or enhance the effects of estrogen respectively. Our objective was to investigate the potential role of COMT in parturition and cervical ripening using a rat model. Immunohistochemistry was conducted to detect and localize the COMT protein in rat uterine tissues during pregnancy. We measured the longitudinal changes in urinary 2-hydroxyestrogen before, during, and after pregnancy in rats. Animal studies were conducted to determine the effect of treatment with a selective COMT inhibitor on (1) mifepristone-induced preterm birth and (2) cervical resistance to stretch in pregnant rats. The intensity of staining for the COMT protein differed within the luminal epithelium, uterine gland epithelium, endometrium, and myometrium during pregnancy. Levels of staining for the COMT protein in rat myometrium were highest on day 1 and lowest on days 8 and 13, but high levels returned by days 16 and 19 of pregnancy. The levels of urinary 2-hydroxyestrogen gradually increased in the first 2 weeks of pregnancy, peaked from days 16 to 18 of pregnancy, and then gradually returned to pre-pregnancy levels after delivery. The percentage of pups retained in the uterus of pregnant rats treated with both mifepristone and COMT inhibitor (48 +/- 15%) was significantly higher (P < 0.05) when compared with the value of pregnant rats treated with mifepristone alone (12 +/- 4%). The resistance to stretch was significantly higher (P < 0.05) in cervical tissues from the pregnant rats treated with COMT inhibitor (0.28) when compared with cervical tissues taken from rats treated with vehicle control (0.18). Modulation of COMT activity may play a role in the regulation of myometrial contractility and cervical ripening during pregnancy.

Synergistic effects of antiprogestins and iNOS or aromatase inhibitors on establishment and maintenance of pregnancy.

Progesterone is known to be involved in many steps in female reproduction including control of implantation and uterine-cervical function during pregnancy. Our studies in rats and guinea pigs indicate that progesterone inhibits uterine contractility and cervical softening during pregnancy. Progesterone levels or actions decline near the end of pregnancy leading to the onset of labor. Treatment with progestin agonists prolongs pregnancy and inhibits cervical softening, whereas treatment with antiprogestins (mifepristone or onapristone) stimulates uterine contractility, cervical softening and premature delivery. Thus the effect of progesterone receptor modulators in the uterus and cervix depend up on the degree of intrinsic agonistic/antagonistic activities. Our recent studies show that progesterone interacts with nitric oxide (NO) to maintain pregnancy and that administration of progesterone antagonists with NO synthase inhibitors act synergistically to stimulate labor. In addition our studies show that combinations of progesterone antagonists with aromatase inhibitors act synergistically to induce labor. Similarly antiprogestins interact with NO synthase or aromatase inhibitors to block implantation through action on the endometrium. These studies suggest new applications for combined therapies of progestin receptor modulators with aromatase inhibitors or agents that modify NO production for contraception, stimulation of labor, estrogen-dependent diseases and improved outcomes in pregnancy.

Platelet-activating factor antagonist WEB-2170 inhibits lipopolysaccharide-induced, but not antiprogestin-induced, preterm cervical ripening in timed-pregnant rats.

OBJECTIVE: The purpose of this study was to determine whether the platelet-activating factor antagonist WEB-2170 inhibits preterm cervical ripening induced by lipopolysaccharide or by antiprogestin RU 486. STUDY DESIGN: Timed-pregnant rats were killed on day 16 after treatment with (1) WEB-2170, lipopolysaccharide, lipopolysaccharide plus WEB-2170, or vehicle control and (2) with WEB-2170, RU 486, RU 486 plus WEB-2170, or vehicle control. Cervical ripening was assessed by light-induced fluorescence and resistance to stretch. Statistics were assessed by 1-way analysis of variance followed by Tukey-test (P <.05). RESULTS: Light-induced fluorescence and resistance to stretch were significantly lower in the lipopolysaccharide-treated and in the RU486-treated animals compared with vehicle control (lipopolysaccharide:light-induced fluorescence, 7.0+/-0.6 vs 12.8+/-0.8 [P=.001]; resistance to stretch, 0.41+/-0.03 N/mm vs 0.54+/-0.04 N/mm [P <.05]; RU486:light-induced fluorescence, 9.6+/-0.6 vs 11.7+/-0.6 [P <.05]; resistance to stretch, 0.28+/-0.06 N/mm vs 0.61+/-0.02 N/mm [P <.001]). Compared with vehicle control, WEB-2170 alone did not alter cervical light-induced fluorescence or resistance to stretch. Although WEB-2170 significantly blocked cervical ripening after lipopolysaccharide administration (light-induced fluorescence, 11.3+/-1.3 [P <.05]; resistance to stretch, 0.61+/-0.04 [P <.01]), WEB-2170 did not inhibit the RU 486-induced cervical ripening. CONCLUSION: Although infection-related cervical ripening is inhibited by platelet-activating factor antagonists, the physiologic process of cervical ripening appears to be unaffected. Platelet-activating factor inhibition may be of clinical value in the infection-related pathologic processes that are responsible for premature cervical ripening.

Local application of platelet-activating factor induces cervical ripening accompanied by infiltration of polymorphonuclear leukocytes in rats.

OBJECTIVE: The purpose of this study was to determine the effect of locally applied platelet-activating factor on cervical ripening and leukocyte infiltration. STUDY DESIGN: Timed-pregnant rats were treated vaginally twice each day on days 14 and 15 of gestation with platelet-activating factor 5 x 10(-8) mol/L (low-dose), 5 x 10(-7) mol/L (high-dose), or solvent and were killed on day 16. Cervical ripening was assessed by cervical resistance-to-stretch and light-induced autofluorescence of cross-linked collagen. Longitudinal sections of the cervix were hematoxylin and eosin stained, and the number of polymorphonuclear leukocytes in eight high-power fields was determined. RESULTS: Cervical resistance-to-stretch and light-induced autofluorescence were decreased in rats that were treated with high-dose platelet-activating factor compared with controls (0.4375 +/- 0.0126 N/mm vs 0.5528 +/- 0.0294 N/mm, and 2.739 +/- 0.150 N/mm vs 3.475 +/- 0.236 N/mm; P <.05). High-dose platelet-activating factor led to infiltration with polymorphonuclear leukocytes compared with control (22.1 +/- 5.2 vs 2.1 +/- 1.3 cells/8 high-power fields, P <.05). CONCLUSION: Local platelet-activating factor application induces cervical ripening accompanied by stromal polymorphonuclear leukocyte infiltration. Platelet-activating factor may play a major role in parturition under physiologic and pathologic conditions.