Silvia Agramunt

Birth Weight, Head Circumference, and Prenatal Exposure to Acrylamide from Maternal Diet: The European Prospective Mother–Child Study (NewGeneris)

Background: Acrylamide is a common dietary exposure that crosses the human placenta. It is classified as a probable human carcinogen, and developmental toxicity has been observed in rodents. Objectives: We examined the associations between prenatal exposure to acrylamide and birth outcomes in a prospective European mother–child study. Methods: Hemoglobin (Hb) adducts of acrylamide and its metabolite glycidamide were measured in cord blood (reflecting cumulated exposure in the last months of pregnancy) from 1,101 singleton pregnant women recruited in Denmark, England, Greece, Norway, and Spain during 2006–2010. Maternal diet was estimated through food-frequency questionnaires. Results: Both acrylamide and glycidamide Hb adducts were associated with a statistically significant reduction in birth weight and head circumference. The estimated difference in birth weight for infants in the highest versus lowest quartile of acrylamide Hb adduct levels after adjusting for gestational age and country was –132 g (95% CI: –207, –56); the corresponding difference for head circumference was –0.33 cm (95% CI: –0.61, –0.06). Findings were similar in infants of nonsmokers, were consistent across countries, and remained after adjustment for factors associated with reduced birth weight. Maternal consumption of foods rich in acrylamide, such as fried potatoes, was associated with cord blood acrylamide adduct levels and with reduced birth weight. Conclusions: Dietary exposure to acrylamide was associated with reduced birth weight and head circumference. Consumption of specific foods during pregnancy was associated with higher acrylamide exposure in utero. If confirmed, these findings suggest that dietary intake of acrylamide should be reduced among pregnant women.

In utero exposure to dioxins and dioxin-like compounds and anogenital distance in newborns and infants

Background: Anogenital distance in animals is used as a measure of fetal androgen action. Prenatal exposure to dioxins and dioxin-like compounds in rodents causes reproductive changes in male offspring and decreases anogenital distance. Objective: We assessed whether in utero exposure to dioxins and dioxin-like compounds adversely influences anogenital distance in newborns and young children (median age, 16 months; range, 1–31 months). Methods: We measured anogenital distance among participants of the “Rhea” mother–child cohort study in Crete and the Hospital del Mar (HMAR) cohort in Barcelona. Anogenital distance (AGD; anus to upper penis), anoscrotal distance (ASD; anus to scrotum), and penis width (PW) were measured in 119 newborn and 239 young boys; anoclitoral (ACD; anus to clitoris) and anofourchetal distance (AFD; anus to fourchette) were measured in 118 newborn and 223 young girls. We estimated plasma dioxin-like activity in maternal blood samples collected at delivery with the Dioxin-Responsive Chemically Activated LUciferase eXpression (DR CALUX®) bioassay. Results: Anogenital distances were sexually dimorphic, being longer in males than females. Plasma dioxin-like activity was negatively associated with AGD in male newborns. The estimated change in AGD per 10 pg CALUX®–toxic equivalent/g lipid increase was –0.44 mm (95% CI: –0.80, –0.08) after adjusting for confounders. Negative but smaller and nonsignificant associations were observed for AGD in young boys. No associations were found in girls. Conclusions: Male infants may be susceptible to endocrine-disrupting effects of dioxins. Our findings are consistent with the experimental animal evidence used by the Food and Agriculture Organization/World Health Organization to set recommendations for human dioxin intake.

Anogenital Distances in Newborns and Children from Spain and Greece: Predictors, Tracking and Reliability

BACKGROUND Anogenital distance has been associated with prenatal exposure to chemicals with anti-androgenic effects. There are limited data in humans concerning descriptive patterns, predictors, and the reliability of measurement of anogenital distances. We examined anogenital distance measurements and their predictors in males and females and further estimated the reliability of these measurements. METHODS Anogenital distances were measured in repeated time periods among 352 newborns and 732 young children in two cohorts, one in Crete, Greece and one in Barcelona, Spain. Mixed effect models were used to estimate the between-children, between- and within-examiners variance, as well as the reliability coefficients. RESULTS Genitalia distances were longer in males than in females. Anogenital distances in both sexes increased rapidly from birth to 12 months, while the additional increase during the second year was small. Birthweight was associated with an increase of 1.9 mm/kg [95% CI 0.1, 3.8] (CI, confidence interval) in the anogenital distance measured from the anus to anterior base of the penis in newborn males, 2.9 mm/kg [95% CI 1.8, 3.9] in anoclitoral distance and 1.0 mm/kg [95% CI 0.0, 2.0] in anofourchettal distance in newborn females, after adjustment for gestational age. In children, body weight was the main predictor of all genitalia measurements. Moreover, anogenital distances at birth were associated with the corresponding distances at early childhood. High reliability coefficients (>90%) were found for all anogenital distances measurements in males and females. CONCLUSIONS Anogenital distances are strongly related to gestational age and birthweight and later, to growth. They track through early life and are highly reliable measures in both sexes.

Bulky DNA Adducts in Cord Blood, Maternal Fruit-and-Vegetable Consumption, and Birth Weight in a European Mother–Child Study (NewGeneris)

Background: Tobacco-smoke, airborne, and dietary exposures to polycyclic aromatic hydrocarbons (PAHs) have been associated with reduced prenatal growth. Evidence from biomarker-based studies of low-exposed populations is limited. Bulky DNA adducts in cord blood reflect the prenatal effective dose to several genotoxic agents including PAHs. Objectives: We estimated the association between bulky DNA adduct levels and birth weight in a multicenter study and examined modification of this association by maternal intake of fruits and vegetables during pregnancy. Methods: Pregnant women from Denmark, England, Greece, Norway, and Spain were recruited in 2006–2010. Adduct levels were measured by the 32P-postlabeling technique in white blood cells from 229 mothers and 612 newborns. Maternal diet was examined through questionnaires. Results: Adduct levels in maternal and cord blood samples were similar and positively correlated (median, 12.1 vs. 11.4 adducts in 108 nucleotides; Spearman rank correlation coefficient = 0.66, p < 0.001). Cord blood adduct levels were negatively associated with birth weight, with an estimated difference in mean birth weight of –129 g (95% CI: –233, –25 g) for infants in the highest versus lowest tertile of adducts. The negative association with birth weight was limited to births in Norway, Denmark, and England, the countries with the lowest adduct levels, and was more pronounced in births to mothers with low intake of fruits and vegetables (–248 g; 95% CI: –405, –92 g) compared with those with high intake (–58 g; 95% CI: –206, 90 g) Conclusions: Maternal exposure to genotoxic agents that induce the formation of bulky DNA adducts may affect intrauterine growth. Maternal fruit and vegetable consumption may be protective. Citation: Pedersen M, Schoket B, Godschalk RW, Wright J, von Stedingk H, Törnqvist M, Sunyer J, Nielsen JK, Merlo DF, Mendez MA, Meltzer HM, Lukács V, Landström A, Kyrtopoulos SA, Kovács K, Knudsen LE, Haugen M, Hardie LJ, Gützkow KB, Fleming S, Fthenou E, Farmer PB, Espinosa A, Chatzi L, Brunborg G, Brady NJ, Botsivali M, Arab K, Anna L, Alexander J, Agramunt S, Kleinjans JC, Segerbäck D, Kogevinas M. 2013. Bulky DNA adducts in cord blood, maternal fruit-and-vegetable consumption, and birth weight in a European mother–child study (NewGeneris). Environ Health Perspect 121:1200–1206; http://dx.doi.org/10.1289/ehp.1206333

Storage conditions and stability of global DNA methylation in placental tissue

AIM The placenta is an informative and easily available tissue for many epidemiological studies. We analyzed the extent to which storage delay affects DNA methylation. MATERIAL & METHODS Biopsies from two placentas were sequentially stored at -80°C after standing at room temperature for 30 min, 1 h, 2 h, 6 h and 24 h. Global DNA methylation was measured by bisulfite pyrosequencing of repetitive elements and the luminometric methylation assay. RESULTS Small changes in global DNA methylation in relation to time-to-storage were observed by pyrosequencing, with a coefficient of variation (COV) of 2.49% (placenta 1) and 2.86% (placenta 2), similar to the mean technical variation observed for pyrosequencing (COV: 1.91 and 1.51%, respectively). A luminometric methylation assay yielded more variable results in the two placentas analyzed, both among time points (COV: 9.13 and 10.35%, respectively) and technical replicates (COV: 11.60 and 9.80%, respectively). CONCLUSION Global DNA methylation is stable at room temperature. However, some techniques to measure methylation might be confounded by DNA degradation caused by a delay in storage.

Maternal diet, prenatal exposure to dioxin-like compounds and birth outcomes in a European prospective mother-child study (NewGeneris)

Maternal diet can result in exposure to environmental contaminants including dioxins which may influence foetal growth. We investigated the association between maternal diet and birth outcomes by defining a dioxin-rich diet. We used validated food frequency questionnaires to assess the diet of pregnant women from Greece, Spain, United Kingdom, Denmark and Norway and estimated plasma dioxin-like activity by the Dioxin-Responsive Chemically Activated LUciferase eXpression (DR-CALUX®) bioassay in 604 maternal blood samples collected at delivery. We applied reduced rank regression to identify a dioxin-rich dietary pattern based on dioxin-like activity (DR-CALUX®) levels in maternal plasma, and calculated a dioxin-diet score as an estimate of adherence to this dietary pattern. In the five country population, dioxin-diet score was characterised by high consumption of red and white meat, lean and fatty fish, low-fat dairy and low consumption of salty snacks and high-fat cheese, during pregnancy. The upper tertile of the dioxin-diet score was associated with a change in birth weight of -121g (95% confidence intervals: -232, -10g) compared to the lower tertile after adjustment for confounders. A small non-significant reduction in gestational age was also observed (-1.4days, 95% CI: -3.8, 1.0days). Our results suggest that maternal diet might contribute to the exposure of the foetus to dioxins and dioxin-like compounds and may be related to reduced birth weight. More studies are needed to develop updated dietary guidelines for women of reproductive age, aiming to the reduction of dietary exposure to persistent organic pollutants as dioxins and dioxin-like compounds.

Feasibility of a new two-step procedure for office hysteroscopic resection of submucous myomas: results of a pilot study

OBJECTIVE To assess the feasibility of a new two-step technique for office hysteroscopic resection of submucous myomas. STUDY DESIGN Between January 2010 and December 2011, all consecutive patients of reproductive age with symptomatic lesions sonographically diagnosed as single mainly intracavitary (G1 or G2) myoma ≤4.0 cm were eligible to participate in a prospective study. They underwent a two-step hysteroscopic procedure, which included preparation of partially intramural myomas with incision of the endometrial mucosa and the pseudocapsule covering the myoma in the first step, and excision of the myoma by means of diode laser four weeks later. All procedures were performed on an outpatient basis and without anesthesia. RESULTS A total of 43 women (mean age 36.7 years) were included. The two-step myomectomy technique was successfully performed in 34 (79.1%) patients. All myomas ≤18 mm were successfully enucleated as compared with 85% of 19-30 mm, and 0% of ≥30 mm (P<0.001). Also, myomas located in the anterior/posterior walls and those located in the fundus/lateral walls were enucleated in 87.9% and 50% of cases, respectively (P=0.020). Success of surgery was not influenced by the initial type of myoma. CONCLUSION The new two-step hysteroscopic myomectomy carried out as an outpatient procedure and without anesthesia is feasible for the excision of symptomatic submucous fibroids.

Micronuclei in cord blood lymphocytes and associations with biomarkers of exposure to carcinogens and hormonally active factors, gene polymorphisms, and gene expression: The newgeneris cohort

Background: Leukemia incidence has increased in recent decades among European children, suggesting that early-life environmental exposures play an important role in disease development. Objectives: We investigated the hypothesis that childhood susceptibility may increase as a result of in utero exposure to carcinogens and hormonally acting factors. Using cord blood samples from the NewGeneris cohort, we examined associations between a range of biomarkers of carcinogen exposure and hormonally acting factors with micronuclei (MN) frequency as a proxy measure of cancer risk. Associations with gene expression and genotype were also explored. Methods: DNA and protein adducts, gene expression profiles, circulating hormonally acting factors, and GWAS (genome-wide association study) data were investigated in relation to genomic damage measured by MN frequency in lymphocytes from 623 newborns enrolled between 2006 and 2010 across Europe. Results: Malondialdehyde DNA adducts (M1dG) were associated with increased MN frequency in binucleated lymphocytes (MNBN), and exposure to androgenic, estrogenic, and dioxin-like compounds was associated with MN frequency in mononucleated lymphocytes (MNMONO), although no monotonic exposure–outcome relationship was observed. Lower frequencies of MNBN were associated with a 1-unit increase expression of PDCD11, LATS2, TRIM13, CD28, SMC1A, IL7R, and NIPBL genes. Gene expression was significantly higher in association with the highest versus lowest category of bulky and M1dG–DNA adducts for five and six genes, respectively. Gene expression levels were significantly lower for 11 genes in association with the highest versus lowest category of plasma AR CALUX® (chemically activated luciferase expression for androgens) (8 genes), ERα CALUX® (for estrogens) (2 genes), and DR CALUX® (for dioxins). Several SNPs (single-nucleotide polymorphisms) on chromosome 11 near FOLH1 significantly modified associations between androgen activity and MNBN frequency. Polymorphisms in EPHX1/2 and CYP2E1 were associated with MNBN. Conclusion: We measured in utero exposure to selected environmental carcinogens and circulating hormonally acting factors and detected associations with MN frequency in newborns circulating T lymphocytes. The results highlight mechanisms that may contribute to carcinogen-induced leukemia and require further research. Citation: Merlo DF, Agramunt S, Anna L, Besselink H, Botsivali M, Brady NJ, Ceppi M, Chatzi L, Chen B, Decordier I, Farmer PB, Fleming S, Fontana V, Försti A, Fthenou E, Gallo F, Georgiadis P, Gmuender H, Godschalk RW, Granum B, Hardie LJ, Hemminki K, Hochstenbach K, Knudsen LE, Kogevinas M, Kovács K, Kyrtopoulos SA, Løvik M, Nielsen JK, Nygaard UC, Pedersen M, Rydberg P, Schoket B, Segerbäck D, Singh R, Sunyer J, Törnqvist M, van Loveren H, van Schooten FJ, Vande Loock K, von Stedingk H, Wright J, Kleinjans JC, Kirsch-Volders M, van Delft JHM, NewGeneris Consortium. 2014. Micronuclei in cord blood lymphocytes and associations with biomarkers of exposure to carcinogens and hormonally active factors, gene polymorphisms, and gene expression: The NewGeneris Cohort. Environ Health Perspect 122:193–200; http://dx.doi.org/10.1289/ehp.1206324

Analysis of perfluoroalkyl substances in cord blood by turbulent flow chromatography coupled to tandem mass spectrometry.

A fast on-line analytical method based on turbulent flow chromatography (TFC) in combination with tandem mass spectrometry has been applied for the first time for the analysis of eighteen perfluoroalkyl substances (PFASs), in cord blood. A simple and rapid sample pre-treatment was optimised consisting on protein precipitation of 100 μL of sample with acetonitrile (1:1) followed by centrifugation during 10 min. The method was adapted to be sensitive enough and robust with minimum sample injection volume requirements (20 μL). The optimised methodology presented method limits of detection (MLOD) between 0.031 and 0.76 μg/L, detection capabilities (CCα) in the range between 0.005 and 0.99 μg/L and decision limits (CCβ) ranging from 0.006 to 1.16 μg/L. The recoveries in blank blood were calculated by spiking experiments with a mixture of 18 PFASs and established between 70 and 126% for most of compounds. Isotopic dilution was carried out for quantification of selected analytes. In-house validation of this new approach was carried out according to the requirements in the 2002/657/EC Decision. Finally the good applicability of this new approach was proved by the analysis of 60 cord blood samples from two different Mediterranean cities, Barcelona (Spain) and Heraklion (Greece). Ions perfluorohexanesulfonate (PFHxS) and perfluorooctanesulfonate (PFOS) were found at highest concentration and the more frequently compounds were PFHxS, PFOS and perfluorooctanoic acid (PFOA). The newly developed method proved to be suitable for large-scale epidemiologic studies, and to the data on PFASs exposure during pregnancy.

In utero exposure to compounds with dioxin-like activity and birth outcomes.

Background: Maternal exposure to dioxins and dioxin-like compounds may affect fetal growth and development. We evaluated the association between in utero dioxin-like activity and birth outcomes in a prospective European mother–child study. Methods: We measured dioxin-like activity in maternal and cord blood plasma samples collected at delivery using the Dioxin-Responsive Chemically Activated LUciferase eXpression (DR CALUX) bioassay in 967 mother–child pairs, in Denmark, Greece, Norway, Spain, and England. Multiple linear regression models were used to investigate the associations with birth weight, gestational age, and head circumference. Results: Plasma dioxin-like activity was higher in maternal sample than in cord samples. Birth weight was lower with medium (−58 g [95% confidence interval (CI) = −176 to 62]) and high (−82 g [−216 to 53]) tertiles of exposure (cord blood) compared with the lowest tertile. Gestational age was shorter by approximately half a week in the highest compared with the lowest (−0.4 weeks [95% CI = −0.8 to −0.1]). This association was stronger in boys than in girls, although the statistical evidence for interaction was weak (P = 0.22). Analysis based on CALUX-toxic equivalents expressed per milliliter of plasma showed similar trends. We found no association between dioxin-like activity in maternal plasma and birth outcomes. Conclusions: Results from this international general population study suggest an association between low-level prenatal dioxin-like activity and shorter gestational age, particularly in boys, with weaker associations for birth weight.