Phd in MOLECULAR MEDICINE

Thesis title: Smoking and alcohol during pregnancy: effects on fetal and child health. The role of oxidative stress and possible dysregulation of neurotrophins.

Introduction: the aim of this study was to evaluate fetal and neonatal effects induced by smoking and alcohol during pregnancy. The role of oxidative stress and possible dysregulation of neurotrophins were also investigated. Material and methods: after written informed consent, we recruited full-term pregnant patients. Exclusion criteria were: age < 20 or > 40 years old; gestational age < 37 weeks + 0 days and > 42 weeks + 0 days; multiple pregnancies; placental pathologies; medically assisted procreation; maternal psychiatric, neurological and/or autoimmune diseases; low socio-economic status, malnutrition, and psychological disorders. Questionnaires were used to investigate smoking and alcohol habits. Ultrasound was performed to evaluate fetal weight, amniotic fluid index, and maternal-fetal Doppler velocimetry. At the time of delivery, we collected a tuft of maternal hair (nicotine, cotinine, ethyl glucuronide-EtG), maternal venous blood and cord blood (reactive oxygen species-ROS, neurotrophins). Placental weight and neonatal data were also collected. Phase I: determination of nicotine, cotinine and EtG on maternal keratin matrix with the gas chromatography-mass spectrometry technique. Positive test for nicotine if > 0.16 ng/mg, cotinine if > 0.07 ng/mg and EtG if > 20 pg/mg. Phase II: Free Oxygen Radicals Test (FORT) and Free Oxygen Radical Defense (FORD) test were used to asses circulating ROS. A condition of oxidative stress was indicated by FORT > 330 Fort Unit and FORD test < 1.08 Trolox equivalents. Phase III: neurotrophins were evaluated on maternal venous blood and cord blood using ELISA. The descriptive statistical analyses were performed using SPSS software (version 29; IBM Corp., Chicago, IL, USA). Student’s t-test was used for independent samples. The study was approved by the ethical committee of Policlinico Umberto I (Rome, Italy) [approval no./ID Prot. 0382/2023]. Results: 119 pregnant patients were enrolled (n=62 were tested for smoking and n=57 for alcohol). Twenty-six patients (42%) out of 62 were active smokers, 18 (29%) were passive smokers, and the remaining 18 (29%) were non-smokers. Three patients (5%) out of 57 resulted alcoholic consumers. The mean neonatal weight was on the 51st percentile for non-smoker patients, on the 49th percentile for passive smokers and on the 8th percentile for active smokers. Comparing active smokers and non-smokers, mean neonatal weight and mean placental weight were significantly lower for active smokers (p=0.0001 and p=0.0060, respectively). Average neonatal weight for alcoholic consumers was below the 2nd percentile. Considering two subgroups (n=10 non-smokers and n=10 active smokers) for ROS determination, Student’s-t test (95% CI, p<0.05) showed a statistically significant higher oxidative stress in the blood of active smoking patients (FORT p<0.0001; FORD p=0.0156). The results on cord blood between active smokers and non-smokers were not statistically significant (FORD test p value = 0.2216). Moreover, FORT and FORD test were negative for all newborns, indicating an optimal condition of oxidative stress and an excellent antioxidant status. Finally, active smoking mothers exhibited significantly lower BDNF levels compared to non-smokers (p = 0.001), whereas their children had significantly higher BDNF levels (p = 0.001). Conclusions: fetal growth restriction was confirmed in the group of active smokers and in patients with alcohol abuse. Smoking induces oxidative stress in pregnant women, while the fetus appears to experience a state of oxidative defense, likely induced by the mother, the mechanisms of which are not yet fully understood. Higher BDNF levels in the offspring of active smoking mothers also suggested potential compensatory mechanisms. The BDNF concentration in cord serum may be a promising indicator of exposure to potential insults occurring in utero, which may result in alterations in neurodevelopmental processes.