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Per- and polyfluoroalkyl substances (PFASs) include persistent organic pollutants whose spread is still ubiquitous. Efforts to substitute substances of high concern with fluorinated alternatives, such as HFPO-DA (GenX), DONA (ADONA), and cC6O4, have been made. The aim of this work was to develop and validate an isotopic dilution liquid chromatography-tandem mass spectrometry (LC–MS/MS) method suitable to quantify 30 PFASs in human plasma. Analytes included legacy PFASs (PFOA, PFOS, and PFHxS), fluorinated alternatives (PFBA, PFBS, 6:2 FTSA, HFPO-DA, DONA, and cC6O4), and newly identified compounds (F-53B and PFECHS). The sample preparation was rapid and consisted of simple protein precipitation and centrifugation. Calibration standards and quality control solutions were prepared with a human pooled plasma containing relatively low background levels of the considered analytes. A complete validation was carried out: the lower limits of quantitation (LLOQs) ranged from 0.009 to 0.245 µg/L; suitable linearity (determination coefficients, R2 0.989–0.999), precision (2.0–19.5%, relative standard deviation), and accuracy (87.9–113.1% of theoretical) were obtained for considered concentration ranges. No significant variations of analyte responses were recorded under investigated storage conditions and during matrix effect tests. The external verification confirmed the accuracy of the method, although limited to 12 analytes. The method was also applied to 38 human plasma samples to confirm its applicability. The developed assay is suitable for large-scale analyses of a wide range of legacy and emerging PFASs in human plasma. To our knowledge, this is the first published method including cC6O4 for human biomonitoring.

Background Per-/polyfluoroalkyl substances (PFASs) are persistent organic pollutants and suspected endocrine disruptors. Objective The aim of this work was to conduct a systematic review with meta-analysis to summarise the associations between prenatal or childhood exposure to PFASs and childhood overweight/obesity. Methods The search was performed on the bibliographic databases PubMed and Embase with text strings containing terms related to prenatal, breastfeeding, childhood, overweight, obesity, and PFASs. Only papers describing a biomonitoring study in pregnant women or in children up to 18 years that assessed body mass index (BMI), waist circumference (WC), or fat mass in children were included. When the estimates of the association between a PFAS and an outcome were reported from at least 3 studies, a meta-analysis was conducted; moreover, to correctly compare the studies, we developed a method to convert the different effect estimates and made them comparable each other. Meta-analyses were performed also stratifying by sex and age, and sensitivity analyses were also performed. Results In total, 484 and 779 articles were retrieved from PubMed and Embase, respectively, resulting in a total of 826 articles after merging duplicates. The papers included in this systematic review were 49: 26 evaluating prenatal exposure to PFASs, 17 childhood exposure, and 6 both. Considering a qualitative evaluation, results were conflicting, with positive, negative, and null associations. 30 papers were included in meta-analyses (19 prenatal, 7 children, and 4 both). Positive associations were evidenced between prenatal PFNA and BMI, between PFOA and BMI in children who were more than 3 years, and between prenatal PFNA and WC. Negative associations were found between prenatal PFOS and BMI in children who were 3 or less years, and between PFHxS and risk of overweight. Relatively more consistent negative associations were evidenced between childhood exposure to three PFASs (PFOA, PFOS, and PFNA) and BMI, in particular PFOS in boys. However, heterogeneity among studies was high. Conclusion Even though heterogeneous across studies, the pooled evidence suggests possible associations, mostly positive, between prenatal exposure to some PFASs and childhood BMI/WC; and relatively stronger evidence for negative associations between childhood exposure to PFASs and childhood BMI.

In women scheduled for cancer treatment, oocytes cryopreservation is a well-established procedure. Random start protocols have been a substantial improvement in this setting, allowing to prevent delay in the initiation of cancer treatments. However, there is still the need to optimize the regimen of ovarian stimulation, to make treatments more patient-friendly and to reduce costs. This retrospective study compares two periods (2019 and 2020), corresponding to two different ovarian stimulation regimens. In 2019, women were treated with corifollitropin, recombinant FSH and GnRH antagonists. Ovulation was triggered with GnRH agonists. In 2020, the policy changed, and women were treated with a progestin-primed ovarian stimulation (PPOS) protocol with human menopausal gonadotropin (hMG) and dual trigger (GnRH agonist and low dose hCG) Continuous data are reported as median [Interquartile Range]. To overcome expected changes in baseline characteristics of the women, the primary outcome was the ratio between the number of mature oocytes retrieved and serum anti-mullerian hormone (AMH) in ng/ml. Overall, 124 women were selected, 46 in 2019 and 78 in 2020. The ratio between the number of mature oocytes retrieved and serum AMH in the first and second period was 4.0 [2.3-7.1] and 4.0 [2.7-6.8], respectively (p = 0.80). The number of scans was 3 [3-4] and 3 [2-3], respectively (p<0.001). The total costs of the drugs used for ovarian stimulation were 940 € [774-1,096 €] and 520 € [434-564 €], respectively (p<0.001). Random start PPOS with hMG and dual trigger represents an easy and affordable ovarian stimulation protocol for fertility preservation in women with cancer, showing similar efficacy and being more friendly and economical.

Pooled quality controls (QCs) are usually implemented within untargeted methods to improve the quality of datasets by removing features either not detected or not reproducible. However, this approach can be limiting in exposomics studies conducted on groups of exposed and nonexposed subjects, as compounds present at low levels only in exposed subjects can be diluted and thus not detected in the pooled QC. The aim of this work is to develop and apply an untargeted workflow for human biomonitoring in urine samples, implementing a novel separated approach for preparing pooled quality controls. An LC-MS/MS workflow was developed and applied to a case study of smoking and non-smoking subjects. Three different pooled quality controls were prepared: mixing an aliquot from every sample (QC-T), only from non-smokers (QC-NS), and only from smokers (QC-S). The feature tables were filtered using QC-T (T-feature list), QC-S, and QC-NS, separately. The last two feature lists were merged (SNS-feature list). A higher number of features was obtained with the SNS-feature list than the T-feature list, resulting in identification of a higher number of biologically significant compounds. The separated pooled QC strategy implemented can improve the nontargeted human biomonitoring for groups of exposed and nonexposed subjects.

Telomere length (TL) is a biomarker of biological aging, with shorter telomeres linked to age-related diseases and cancers. Benzene, a known carcinogen, may contribute to telomere shortening through oxidative DNA damage and other mechanisms. Previous studies gave conflicting results. The aim of this study is to investigate the association between benzene and TL in a study population characterized by a wide range of mostly low exposure levels. We enrolled 423 workers occupationally exposed to benzene and 190 non-occupationally exposed referents from three cities in Italy and one in Bulgaria. Participants wore passive and active samplers for an entire work shift to measure time-weighted average benzene exposure. TL was measured through real-time PCR. We applied multivariable mixed-effects models with a random intercept on the city, adjusted for sex, age, smoking, cigarettes/day, and alcohol consumption to evaluate the association between benzene and changes in TL. Exposure to environmental benzene ranged from 0.0013 to 21.14 ppm (min-max). For every ten-fold increase in benzene concentrations, TL decreased by 7.4% (95% confidence interval [CI]: -9.9; -4.7). A similar pattern was observed in never-smokers (-10.2%, 95%CI: -14.9; -5.3) and after additional adjustment for toluene (-6.5%, 95%CI: -9.7; -3.2). When data were available, we observed a negative association also between TL and urinary benzene (-5.8%, 95%CI: -8.7; -2.8 for each twofold increase in urinary benzene concentration). Our findings suggest that even low levels of benzene exposure may cause telomere shortening and accelerate biological aging.

Background Tobacco smoking is a leading cause of morbidity and mortality worldwide, responsible for about 61,300 deaths annually in Italy. In addition to tobacco cigarettes, the use of electronic cigarettes (e-cigs) and heated tobacco products (HTPs) has increased among young people, raising public health concerns. This study aimed to investigate smoking habits, exposure to passive smoke, and awareness of health risks among Italian university students. Methods A multicentre observational survey was carried out by 8 Italian Universities to promote smoke-free lifestyles among students. All students were sent an anonymous online questionnaire via email regarding their smoking habits, their exposure to second-hand smoke, and their knowledge of, and opinions on, smoking. Descriptive statistics and multinomial logistic regression models were applied to assess associations between tobacco and nicotine use with sex and areas of study. Results A total of 20,644 students from different study areas were included in the study: 62.3% did not smoke, 9.9% were former smokers, 14.4% were smokers of tobacco cigarettes exclusively, 6.5% of e-cigs or HTPs, and 6.9% were dual smokers (total smokers 27.8%). Smoking prevalence was highest among Law (35.9%) and Economics (33.7%) students, and lowest among Medicine and Sciences and Technology students (22%). Males were more likely to smoke tobacco or to be dual users, while females reported higher e-cig use; most smokers consumed fewer than 10 cigarettes/day with mild nicotine dependence. Among e-cig/HTP users, over 60% started as an alternative to cigarettes, but 22% started or resumed tobacco smoking after using them and 22% had never smoked tobacco cigarettes before. Passive smoke exposure was common: 26.5% lived with smokers and 58.9% reported peer exposure. While nearly all students recognized the harms of both active and passive tobacco smoking, only 60% considered passive exposure to e-cigs/HTPs as dangerous. About 70% were favourable to stricter enforcement of university smoking bans. Conclusions: Traditional cigarettes remain the most used product among Italian university students, but e-cigs and HTPs are increasingly widespread and socially accepted, even among never smokers. Awareness of the risks of alternative products is limited, and passive exposure remains underestimated. These findings underline the importance of targeted educational campaigns and stronger enforcement of smoke-free policies in academic settings.

Background Pregnancy involves a fine-tuned hormonal interplay between the fetus, placenta, and mother, which shapes long-term developmental outcomes. Endometriosis has been hypothesized to originate in utero due to altered fetal exposure to sex steroids. This study investigates differences in umbilical cord estradiol and androgen levels in female fetuses born to women with and without endometriosis, exploring a potential role of altered in utero hormone exposure in the intergenerational transmission of endometriosis risk. Methods This is a case-control study nested within a cohort of women delivering singleton females at our institution between June 2022 and June 2023. Cases were women with endometriosis (diagnosed via imaging or surgery before pregnancy) and controls were women without endometriosis. Analyses were performed before and after propensity-score matching (PSM) at a 1:3 ratio to control for maternal age, gestational age, and delivery mode. Umbilical cord blood was collected at delivery, and hormonal levels of corticosteroids, progestins, androgens, and estradiol (E2) were measured using liquid chromatography-tandem mass spectrometry. Estradiol-to-androgens ratios were calculated, with 95% confidence intervals (CIs) determined using the bootstrapping method. Results The total cohort included 160 women, 17 (10.6%) of whom had endometriosis. After 1:3 matching, 51 controls without endometriosis were included. Women with endometriosis had higher E2 levels compared to controls, both before PSM (6.8 [4.3–9.1] mcg/L vs. 3.6 [1.4–8.6] mcg/L, p  = 0.03) and after PSM (2.4 [1.1–6.6] mcg/L, p  = 0.002). Estradiol-to-androgens ratios indicated a higher-estrogen and lower-androgens hormonal status in endometriosis, with higher E2-to-testosterone ( p  < 0.001), E2-to-androstenedione ( p  = 0.001), E2-to-dehydroepiandrosterone (DHEA) ( p  < 0.001), and E2-to-DHEA sulfate (DHEAS) ratios ( p  < 0.001) compared to controls. After PSM, the E2-to-testosterone, E2-to-androstenedione, E2-to-DHEA, and E2-to-DHEAS ratios were 4.38 (95% CI: 2.28–6.49), 3.28 (95% CI: 1.29–5.28), 2.52 (95% CI: 1.32–3.72), and 4.57 (95% CI: 1.86–7.27) times higher, respectively. Conclusions This is the first study showing that female newborns of women with endometriosis are exposed to higher in utero estradiol compared to controls. This high estrogen low androgens environment may influence fetal programming of reproductive traits and might drive the in utero susceptibility to endometriosis.

Tebuconazole (TEB) is a fungicide widely used in vineyards and is a suspected teratogen for humans. The aim of this research was to identify urinary biomarkers and the best sampling time for the biological monitoring of exposure to TEB in agricultural workers. Seven vineyard workers of the Monferrato region, Piedemont, Italy, were investigated for a total of 12 workdays. They treated the vineyards with TEB for 1–2 consecutive days, one of them for 3 days. During each application coveralls, underwears, hand washing liquids and head coverings were used to estimate dermal exposure. For biomonitoring, spot samples of urine from each individual were collected starting from 24 h before the first application, continuing during the application, and again after the application for about 48 h. TEB and its metabolites TEB-OH and TEB-COOH were measured by liquid chromatography/triple quadrupole mass spectrometry. TEB contamination of coveralls and total dermal exposure showed median levels of 6180 and 1020  μ g. Urinary TEB-OH was the most abundant metabolite; its excretion rate peaked within 24 h after product application (post 24 h). In this time frame, median levels of TEB-OH and TEB-COOH ranged from 8.0 to 387.8  μ g/l and from 5.7 to 102.9  μ g/l, respectively, with a ratio between the two metabolites of about 3.5. The total amount of urinary metabolites (U-TEB eq ) post 24 h was significantly correlated with both TEB on coveralls and total dermal exposure (Pearson’s r =0.756 and 0.577). The amount of metabolites excreted in urine represented about 17% of total dermal TEB exposure. Our results suggest that TEB-OH and TEB-COOH in post-exposure urine samples are promising candidates for biomonitoring TEB exposure in agricultural workers.

Glyphosate-based herbicides are the most widely used pesticides in the world; however, the toxicity of glyphosate (GlyP) toward humans, especially its carcinogenicity, is controversial. The aim of this work was to validate a rapid assay for measuring GlyP and its metabolite aminomethylphosphonic acid (AMPA) in urine for human biomonitoring. The analytes were purified via solid-phase extraction in the presence of isotopically labeled internal standards. An LC-MS/MS assay was developed using a column with a novel hybrid stationary phase combined with anion exchange and hydrophilic interaction liquid chromatography. Detection and quantification were performed using negative electrospray ionization in a hybrid triple quadrupole/linear ion trap mass spectrometer. The retention times for AMPA and GlyP were 1.44 and 7.24 min, respectively. Calibration curves showed a linear dynamic range of up to 40 µg/L, inter- and intra-run precisions <7.5%, and accuracies within 10% of the theoretical concentrations. The limits of quantification were 0.1 µg/L and 0.5 µg/L for GlyP and AMPA, respectively. The matrix effect bias was controlled using internal standards. Successful participation in external quality assurance exercises strengthens the validity of the method. The assay was applied to the measurement of GlyP and AMPA in the urine of 9 urban residents, 26 rural residents, and 12 agricultural workers; while AMPA was mostly not quantifiable, the median GlyP values were 0.1 and 0.34 µg/L in rural residents and workers, respectively. The assay is useful to assess GlyP and AMPA in human urine following different exposure scenarios.