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.
Smoking has long been associated with adverse perinatal outcomes that include fetal growth restriction, premature rupture of membranes, and preterm birth. Simpson in 1957 was the first to report the association between smoking and preterm births. It has been recognized since then that smoking is a high risk factor of preterm births. Nicotine is the major active agent among >4000 toxic constituents of cigarette smoke and tobacco. The effects of nicotine exposure on pregnancy outcomes during pregnancy have received far less attention with few publications, and the results are inconsistent. Gaither et al in 2009 also reported that nicotine use increased preterm births. However, results from rat studies indicate that nicotine prolongs gestation. Results from a nicotine patch replacement trial in humans also show that nicotine exposure increased the cesarean delivery rate, which suggests delayed cervical ripening. At the cellular level, nicotine could induce the synthesis of type I collagen because some studies indicate that nicotine exposure increases dermal collagen cross-links expression. Nicotine is an antiinflammatory agent that is mediated by cholinergic antiinflammatory pathways, which has been described in numerous reports. Nicotine suppresses inflammation during experimental ulcerative colitis, attenuates macrophage infiltration in rat lung, and blocks leukocyte recruitment in vivo.
Cervical ripening refers to a 3-state process of softening, effacement, and dilation that begins before the onset of labor contractions. This process is necessary for normal birth because it allows the fetus to pass through the cervix and vagina. Failure or disruption of cervical ripening causes many obstetric complications. Premature cervical ripening can lead to preterm birth, which occurs at an alarming rate of 12.7% of all births in the United States, although failure of cervix ripening during pregnancy often leads to drug treatment and induction or even cesarean delivery. The process of cervical ripening is influenced greatly by the cervical stroma, which is composed primarily of collagen and stroma cells that consists of fibroblasts and, to a lesser extent, smooth muscle cells. These stroma cells secrete an extracellular matrix that is rich in fibrillar collagen, matricellular proteins, elastin, glycosaminoglycans, and proteoglycans. Changes in the extracellular matrix composition over the course of pregnancy and labor determine the flexibility and mechanical strength of the cervix. Akins et al recently observed that collagen content remains constant throughout pregnancy and parturition but that the cervical cross-links were reduced during the process of cervical ripening. Studies of the light-induced fluorescence of cervical collagen also indicate that cross-links progressively decrease during gestation and reach the lowest levels during parturition. Thus, the decrease of cervical collagen cross-links influences the solubility of collagen during the process of cervical ripening. The process of parturition and cervix ripening has been likened to an inflammatory reaction with influx of neutrophils and macrophages into the cervix and an increased cervical level of proinflammatory cytokines such as interleukin-8. These proinflammatory cytokines increase the secretion of collagenase and elastase, which is critical for cervical collagen degradation.
Cervical ripening has been qualified biochemically by assessment of the various cervical microstructure components, physically by measurement of the extensibility of isolated cervical tissue, and clinically by digital examination or ultrasound scanning. Recently, the light-induced fluorescence (LIF) of collagen to estimate changes in the cervix has been used in several animal models. The LIF approach that measures the collagen cross-links of the cervix is a novel and noninvasive method.
Based on the observations that nicotine prolongs gestation and collagen metabolism is regulated by nicotine, we hypothesize that nicotine could have many possible side-effects on pregnancy such as an inhibition of inflammation-associated birth and a delay in delivery that is caused by altered cervical function and/or uterine contractility. The present study was designed to elucidate the possible mechanism of nicotine’s ability to prolong gestation and its effects on the cervix and myometrium. Our results demonstrate that nicotine suppresses the normal physiologic decrease of cervical collagen cross-links that occurs during gestation and thereby inhibits cervical ripening and consequently delays delivery without an effect on the myometrium to influence contractility.
Materials and Methods
Animals
Timed-pregnant Sprague-Dawley rats (240-280 g) from Charles-River Laboratories (Wilmington, MA) were delivered to our animal care facilities on day 13 of gestation (day 1 being the day when a sperm plug was observed). The rats normally deliver on the morning of day 22 of gestation. The animals were housed separately, with free access to food and water, and were maintained on a constant 12-hour light-dark cycle. 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 rats were allocated randomly to control group and nicotine groups. The rats were killed by C o 2 inhalation after delivery. All procedures were approved by the Animal Care and Use Committee of the St. Joseph’s Hospital and Medical Center in Phoenix.
Reagents
Nicotine hydrogen tartrate (Sigma Chemical Co, St. Louis, MO) was dissolved in a vehicle that consisted of sterile 0.9% saline solution and was adjusted to pH 7.1-7.3 with NaOH. A stock solution of 1 mg/mL nicotine (expressed as base) was frozen in aliquots, and 1 aliquot was thawed to prepare fresh drug solution each day. Control solutions for injection consisted of vehicle.
Treatment
Rats in the nicotine groups were injected subcutaneously with 0.25, 0.5, 1, or 2 mg nicotine/kg of body weight twice a day (9:00 am and 3:00 pm ) from day 14 of pregnancy until day 22. Control rats received an equivalent volume of physiologic saline solution (0.9% NaCl) at comparable times. Measurements of cervical LIF were performed in the animals every other day, starting at day 14 of gestation until day 20. For the cervical to resistance to stretch studies, rats were killed on day 20 of gestation; pups and placentas were also removed and weighed to confirm the influence of nicotine on fetal growth. Times of delivery of controls and various nicotine groups were recorded. The expulsion of 1 pup was defined as delivery. Blood was collected on days 14, 17, and 20 of gestation; the plasma progesterone levels were analyzed by a specific chemiluminescent microparticle immunoassay (Abbott Laboratories, Abbott Park, IL).
Cervical collagen measurements
The amount of cervical collagen was evaluated in vivo by the measurement of the autofluorescent properties of cross-linked collagen with a collascope (Reproductive Research Technologies, Houston, TX) as previously described. Briefly, 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 of fluorescence that are emitted by the collagen of 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 rat at any given time.
Measurements of cervical distensibility
The rats were treated with nicotine (1 mg/kg in 0.3 mL NaCl, subcutaneously) or NaCl (control group, 0.3 mL) twice a day from day 14 of gestation until day 20. Cervices were then removed for the cervical resistance test. The cervix was defined as the least vascular tissue with 2 parallel lumina between the uterine horns and the vagina. Connective tissue and fat were removed, and the cervix was suspended with its longitudinal axis vertically in a 10-mL organ chamber for isometric tension recording. The chambers were filled with physiologic Krebs solution, bubbled with a mixture of 95% O 2 /5% C o 2 , and maintained at 37°C. After hooks were inserted through each of the canals, 1 side of the cervix was fixed to the bottom of the organ chamber; the other side was connected to a force transducer that, in turn, was connected to an online computer. The tissues were mounted for isometric recording under 1 g tension for 1 hour. The isolated cervix was then stretched incrementally in steps of 0.5 mm at 4-minute intervals; the tension was recorded continuously. The resultant length-tension curve had a saw-tooth appearance. The slope of the regression line through the linear portion of the length-tension curve was derived and used as an indication of cervical extensibility. The slope of the length-tension curve is related directly to cervical resistance and inversely to compliance. The more resistant and less compliant the cervix is, the steeper is the change in tension in response to each increment in length.
Uterine contractility measurements
Timed-pregnant rats were killed by C o 2 inhalation on day 16 of gestation. After the uterus was removed, myometrial strips (width, 2 mm; length, 10 mm) were mounted for isometric recording with <1 g of tension in organ baths as previously described. The tissue bath contained 10 mL of Krebs-Henseleit physiologic salt solution that was maintained at 37°C, pH 7.4, bubbled with a mixture of 95% O 2 /5% C o 2 . Myometrial strips were allowed to equilibrate for at least 1 hour during which time the Krebs-Henseleit physiologic salt solution was changed every 20 minutes. After equilibration, a 30-minute period was allowed to achieve spontaneous phasic contractions. The nicotine was then added to the tissue bath in a cumulative manner at bath concentrations of 0.1, 1, 10, and 100 nmol/L and 1, 10, and 100 μmol/L at 30-minute intervals. The control tissue experiments were performed in the following manner: comparative volumes of saline solution were added to the tissue bath at 30-minute intervals. The effects of nicotine and control solutions were assessed by estimates of the dose/response relationship, calculated as the integral of selected areas under the contraction curves for each 30-minute interval, and expressed as a percentage of the integral that was obtained 30 minutes before any nicotine addition with the use of a PowerLab hardware unit (ADInstruments, Colorado Springs, CO) and Chart software (version 7.0; ADInstruments).
Statistical analysis
Results are expressed as means ± SEM. Data analysis between 2 groups were performed with an unpaired Student t test and between multiple groups with a 1-way analysis of variance followed by the Student-Newman-Keuls test. Data were analyzed for statistical significance with SigmaStat software (version 3.01A for Windows; Systat Software Inc, Richmond, CA). Statistical significance was defined as a probability value of < .05.
Results
Effects of nicotine treatments on rat delivery time and plasma progesterone
The gestation length in the higher nicotine dose groups (1 and 2 mg/kg/d) is significantly longer ( P = .002) compared with the control group ( Figure 1 , A). The lower doses of nicotine also appear to prolong the gestation, although there are no significant differences compared with the control group ( P = .62; n = 5).
The plasma progesterone values for rats of a nicotine group (1 mg/kg/d) and a control group of pregnant rats on day 20 of gestation were 94.8 ± 5.3 ng/mL and 86.1 ± 7.2 ng/mL, respectively. There was no significant difference ( P = .34). Similarly, plasma progesterone values were not significantly different ( P = .28 and .39, respectively) between control and nicotine-treated rats (n = 6) on days 14 and 17 of gestation (data not presented).
Effects of nicotine on rat pup and placenta weights
For the pup and placenta weights, there are no significant differences between the nicotine-treated rats (n = 6 for each group) and the control rats on day 20 of gestation ( Figure 1 , B and C, with probability values of .067 and .094, respectively).
Effects of nicotine on cervical LIF
The effect of nicotine on cervical LIF ( Figure 2 ) followed a positive dose-response pattern: The higher dose (1 and 2 mg/kg/d) resulted in a significantly higher LIF ( P < .001; n = 6); with lower doses (0.25 and 0.5 mg/kg/d), there are no significant differences compared with the control group ( P = .12).
