Using a prospective cohort design study, 30 pregnant smokers and 34 pregnant non-smokers were recruited between September 2008 and September 2009 from the Low Risk Prenatal Care Center of the Obstetric Department at the Hospital Sao Lucas da Pontificia Universidade Catolica do Rio Grande do Sul (HSL-PUCRS) in Porto Alegre, Brazil.
The inclusion criteria were: pregnant women between 18 and 35 years of age, in the third trimester of gestation (dated in the first trimester), regularly receiving prenatal care, had no gestational complaints and agreed to participate in the study by signing the consent form. The exclusion criteria were: ultrasonographic diagnosis of fetal malformations, clinical situations related to the pregnancy or any other disease that could interfere with fetal growth, illiteracy, psychiatric disorders, alcoholism, former smokers and the possibility to move to another city and to lose contact during the study. Patients who discontinued prenatal care and those that stopped or started smoking during their pregnancy were also excluded. No patient was excluded after inclusion in the study because no patient discontinued the follow up during the prenatal period.
Smoking was determined according to the report of using cigarettes from the periconceptional period to the day of the interview. The periconceptional period was defined as the period starting one month before the date of the last menstruation. To be considered a non-smoker, patients must have stated that they had never smoked, especially during their pregnancy, and reports were confirmed by a biochemical marker.
All patients were submitted to same tests to confirm their smoking status: concentration of carbon monoxide in the exhaled air and concentration of urinary cotinine. We found no discrepancy, that is, no patient who said was a non-smoker was detected as a smoker.
At the initial medical visit, the objectives of the study were explained and the consent form signature was obtained.
Next, a standardized questionnaire was completed, pregnancy and smoking data were collected and the COex measurement was performed. A urine sample was also collected to measure cotinine levels, and an ultrasonograph examination was performed. Smoking between the tests was not permitted. After the birth, patient was contacted to collect data about the newborn one month after delivery.
Doppler velocimetry exam with colored mapping of the uterine arteries, umbilical arteries and fetal medium cerebral arteries was performed with an Ultrasonix machine (Sonix Model, Version 2.5, Ultrasonix Medical Corporation, Canada), and the resistance indices (RI) were recorded. We also recorded the amniotic fluid index, the placental grade, fetal biometry and the estimated fetal weight placed on a percentile curve for the gestational age to adjust for possible bias.
To decrease possible bias at ultrasonography due to a large variability of Doppler values, three measurements were taken, and the mean was recorded.
COex was measured by a MicroCO Meter (MicroCO - Micro Medical Ltd., Rochester, Kent, UK) using an electrochemical sensor. Patients were instructed to breathe deeply, hold their breath for 20 seconds and then to exhale slowly and completely through a mouthpiece. Smokers had COex measured no more than two hours after their last smoked cigarette .
For measurement of the cotinine concentration in the urine, a previously-validated high performance liquid chromatography (HPLC) method was used .
Whereas the ultrasonography examinations and the exhaled CO measurements were performed by the same author (JBDM), the cotinine measurements were performed at the PUCRS Toxicology Institute by a technician blind to patient study group.
Mean ± SD were used to describe the symmetric, continuous data and the median and range was used for asymmetric data. A percentile calculation was used for categorical data. Student's t test and ANOVA were used to compare means. Adjustments for potential confounding effects were made by covariance analysis (ANCOVA) and multiple linear regression. Possible confounding factors included in the analysis for adjustment were patient age, gestational age, degree of placenta, amniotic fluid index, number of pregnancies. The assigned level of significance was 0.05. The data were processed by the Statistical Package for the Social Sciences (SPSS) Version 17.0
Patients were divided into four groups according to the levels of COex (Group1: 0 ppm; Group 2: 1-4 ppm; Group 3: 5-9 ppm; Group 4: ≥10 ppm) in order to analyze possible changes in the resistance indices. The same analyses were also performed according to the levels of urinary cotinine (Group 1: 0 ng/mL; Group 2: <50 ng/mL; Group 3: 50-500 ng/mL; Group 4 >500 ng/mL).
We used percentiles to compare the resistance indices and fetal birth weights according to the gestational age of each patient. For weight, a Hadlock table was used . Analysis of the resistance index values was performed by using the published standards for the respective percentiles .
The study was approved by the PUCRS Research Ethics Committee, under No. 08/04319 on May 09th 2008.