Danuta Ligocka

Human urinary phthalate metabolites level and main semen parameters, sperm chromatin structure, sperm aneuploidy and reproductive hormones

The aim of the study was to assess the association of phthalate metabolites levels in urine with semen parameters (sperm concentration, motility, morphology, CASA parameters), sperm chromatin structure, sperm aneuploidy and reproductive hormones. The study population consisted of 269 men who were attending an infertility clinic and had normal semen concentration (20-300mln/ml) or slight oligozoospermia (15-20mln/ml). Participants were interviewed and provided a semen sample. The phthalate metabolites were analysed in the urine using a procedure based on the LC-MS/MS method. Urinary phthalate metabolites levels were significantly associated with a decrease in sperm motility (5OH MEHP, MEHP, MINP), CASA parameters (MBP), testosterone level (MEHP) and an increase sperm DNA damage (MBP) and sperm aneuploidy (MBzP, MBP, MEHP, MEP). In view of the importance of human reproductive health and the widespread usage of phthalates, it is important to further investigate these correlations.

First steps toward harmonized human biomonitoring in Europe: demonstration project to perform human biomonitoring on a European scale.

Background For Europe as a whole, data on internal exposure to environmental chemicals do not yet exist. Characterization of the internal individual chemical environment is expected to enhance understanding of the environmental threats to health. Objectives We developed and applied a harmonized protocol to collect comparable human biomonitoring data all over Europe. Methods In 17 European countries, we measured mercury in hair and cotinine, phthalate metabolites, and cadmium in urine of 1,844 children (5–11 years of age) and their mothers. Specimens were collected over a 5-month period in 2011–2012. We obtained information on personal characteristics, environment, and lifestyle. We used the resulting database to compare concentrations of exposure biomarkers within Europe, to identify determinants of exposure, and to compare exposure biomarkers with health-based guidelines. Results Biomarker concentrations showed a wide variability in the European population. However, levels in children and mothers were highly correlated. Most biomarker concentrations were below the health-based guidance values. Conclusions We have taken the first steps to assess personal chemical exposures in Europe as a whole. Key success factors were the harmonized protocol development, intensive training and capacity building for field work, chemical analysis and communication, as well as stringent quality control programs for chemical and data analysis. Our project demonstrates the feasibility of a Europe-wide human biomonitoring framework to support the decision-making process of environmental measures to protect public health. Citation Den Hond E, Govarts E, Willems H, Smolders R, Casteleyn L, Kolossa-Gehring M, Schwedler G, Seiwert M, Fiddicke U, Castaño A, Esteban M, Angerer J, Koch HM, Schindler BK, Sepai O, Exley K, Bloemen L, Horvat M, Knudsen LE, Joas A, Joas R, Biot P, Aerts D, Koppen G, Katsonouri A, Hadjipanayis A, Krskova A, Maly M, Mørck TA, Rudnai P, Kozepesy S, Mulcahy M, Mannion R, Gutleb AC, Fischer ME, Ligocka D, Jakubowski M, Reis MF, Namorado S, Gurzau AE, Lupsa IR, Halzlova K, Jajcaj M, Mazej D, Snoj Tratnik J, López A, Lopez E, Berglund M, Larsson K, Lehmann A, Crettaz P, Schoeters G. 2015. First steps toward harmonized human biomonitoring in Europe: demonstration project to perform human biomonitoring on a European scale. Environ Health Perspect 123:255–263; http://dx.doi.org/10.1289/ehp.1408616

Fish consumption patterns and hair mercury levels in children and their mothers in 17 EU countries

The toxicity of methylmercury (MeHg) in humans is well established and the main source of exposure is via the consumption of large marine fish and mammals. Of particular concern are the potential neurodevelopmental effects of early life exposure to low-levels of MeHg. Therefore, it is important that pregnant women, children and women of childbearing age are, as far as possible, protected from MeHg exposure. Within the European project DEMOCOPHES, we have analyzed mercury (Hg) in hair in 1799 mother-child pairs from 17 European countries using a strictly harmonized protocol for mercury analysis. Parallel, harmonized questionnaires on dietary habits provided information on consumption patterns of fish and marine products. After hierarchical cluster analysis of consumption habits of the mother-child pairs, the DEMOCOPHES cohort can be classified into two branches of approximately similar size: one with high fish consumption (H) and another with low consumption (L). All countries have representatives in both branches, but Belgium, Denmark, Spain, Portugal and Sweden have twice as many or more mother-child pairs in H than in L. For Switzerland, Czech Republic, Hungary, Poland, Romania, Slovenia and Slovakia the situation is the opposite, with more representatives in L than H. There is a strong correlation (r=0.72) in hair mercury concentration between the mother and child in the same family, which indicates that they have a similar exposure situation. The clustering of mother-child pairs on basis of their fish consumption revealed some interesting patterns. One is that for the same sea fish consumption, other food items of marine origin, like seafood products or shellfish, contribute significantly to the mercury levels in hair. We conclude that additional studies are needed to assess and quantify exposure to mercury from seafood products, in particular. The cluster analysis also showed that 95% of mothers who consume once per week fish only, and no other marine products, have mercury levels 0.55 μg/g. Thus, the 95th percentile of the distribution in this group is only around half the US-EPA recommended threshold of 1 μg/g mercury in hair. Consumption of freshwater fish played a minor role in contributing to mercury exposure in the studied cohort. The DEMOCOPHES data shows that there are significant differences in MeHg exposure across the EU and that exposure is highly correlated with consumption of fish and marine products. Fish and marine products are key components of a healthy human diet and are important both traditionally and culturally in many parts of Europe. Therefore, the communication of the potential risks of mercury exposure needs to be carefully balanced to take into account traditional and cultural values as well as the potential health benefits from fish consumption. European harmonized human biomonitoring programs provide an additional dimension to national HMB programs and can assist national authorities to tailor mitigation and adaptation strategies (dietary advice, risk communication, etc.) to their countrys specific requirements.

Lifestyle and semen quality: role of modifiable risk factors

Abstract The relationship between exposure to lifestyle factors and adverse effects on human reproductive health is debated in the scientific literature and these controversies have increased public and regulatory attention. The aim of the study was to examine the association between modifiable lifestyle factors and main semen parameters, sperm morphology, and sperm chromatin structure. The study population consisted of 344 men who were attending an infertility clinic for diagnostic purposes with normal semen concentration of 20–300 M/ml or with slight oligozoospermia (semen total concentration of 15–20 M/ml) [WHO 1999]. Participants were interviewed and provided semen samples. The interview included questions about demographics, socio-economic status, medical history, lifestyle factors (consumption of alcohol, tobacco, coffee intake, cell phone and sauna usage), and physical activity. The results of the study suggest that lifestyle factors may affect semen quality. A negative association was found between increased body mass index (BMI) and semen volume (p = 0.03). Leisure time activity was positively associated with sperm concentration (p = 0.04) and coffee drinking with the percentage of motile sperm cells, and the percentage of sperm head and neck abnormalities (p = 0.01, p = 0.05, and p = 0.03, respectively). Drinking red wine 1–3 times per week was negatively related to sperm neck abnormalities (p = 0.01). Additionally, using a cell phone more than 10 years decreased the percentage of motile sperm cells (p = 0.02). Men who wore boxer shorts had a lower percentage of sperm neck abnormalities (p = 0.002) and percentage of sperm with DNA damage (p = 0.02). These findings may have important implications for semen quality and lifestyle.

Phthalate exposure and child development: the Polish Mother and Child Cohort Study.

BACKGROUND Widespread phthalate exposure has prompted investigations concerning their potential adverse health effects. AIM The objective of this study was to evaluate the impact of pre and early postnatal phthalate exposure on child psychomotor development based on the data from the prospective Polish Mother and Child Cohort Study (REPRO PL). STUDY DESIGN, SUBJECTS AND OUTCOME MEASURES Phthalate exposure was determined by measuring 11 phthalate metabolites (MEP, MiBP, MnBP, 3OH-MnBP, MBzP, MEHP, 5OH-MEHP, 5oxo-MEHP, OH-MiNP, oxo-MiNP, and MnOP) in the urine collected from mothers during the third trimester of pregnancy (prenatal exposure) and from their children at the 24th month of age (postnatal exposure). The analysis was performed by the HPLC-MS/MS method. Psychomotor development was assessed in children at the age of 2years by the Bayley Scales of Infant and Toddler Development. RESULTS Child motor development was inversely associated with natural log concentrations (μg/g creatinine) of 3OH-MnBP (β= -2.3; 95% CI -4.0 to -0.6), 5OH-MEHP (β= -1.2; 95% CI -2.2 to -0.3), 5oxo-MEHP (β= -1.8; 95% CI -3.3 to -0.2) and sum of DEHP metabolites (β= -2.2; 95% CI -3.6 to -0.8), DnBP metabolites (β= -1.9; 95% CI -3.4 to -0.4), and high molecular weight phthalates (β= -2.5; 95% CI -4.1 to -0.9) in the urine collected from mothers during pregnancy after adjustment for a variety of potential confounders. Additional adjustment for postnatal phthalate exposure did not change the results. Postnatal child exposure to phthalates was not associated with any of the measured scores of child psychomotor development. CONCLUSIONS The study findings add further support to the possibility that prenatal phthalate exposure may be detrimental to child neurodevelopment and underscore the importance of policies and public health interventions aiming at reduction of such exposure.

Polish mother and child cohort study — defining the problem, the aim of the study and methodological assumptions

OBJECTIVES Exposures during prenatal period have implications for pregnancy outcome as well as for childrens health, morbidity and mortality. Prospective cohort study design allows for the identification of exposures that may influence pregnancy outcome and childrens health, verification of such exposures by biomarker measurements and notification of any changes in exposure level. MATERIALS AND METHODS Polish Mother and Child Cohort Study (REPRO_PL) is multicenter prospective cohort study conducted in 8 different regions of Poland. The final cohort is intended to comprise 1300 mother-child pairs to be recruited within 4-year period (2007-2011). The recruitment and all scheduled visits are conducted in maternity units or clinics in the districts included in the study. The women are followed-up 3 times in pregnancy (once in each trimester) and after delivery for the notification of pregnancy outcome. During each visit, detailed questionnaire and biological samples are collected including saliva, urine, hair, maternal blood and cord blood. About 6 weeks postpartum, breast milk from part of the women is collected. The study concentrates on the identification and evaluation of the effects of prenatal environmental exposure on pregnancy outcome and childrens health. Specific research hypotheses refer to the role of heavy metals, exposure to polycyclic aromatic hydrocarbons (PAHs) and environmental tobacco smoke (ETS) in the aetiology of small-for-gestational-age (SGA) and preterm delivery (PD). The role of oxidative stress putative mechanism and pregnant women nutritional status will be investigated. Based on questionnaire data, the impact of occupational exposures and stressful situations will be evaluated. RESULTS The results of the study will become available within the next few years and will help to determine levels of child prenatal exposure in several areas of Poland and its impact on course and outcome of pregnancy.

Estimation of Cutoff Values of Cotinine in Urine and Saliva for Pregnant Women in Poland

Setting appropriate cutoff values and the use of a highly sensitive analytical method allow for correct classification of the smoking status. Urine-saliva pairs samples of pregnant women in the second and third trimester, and saliva only in the first trimester were collected. Offline SPE and LC-ESI-MS/MS method was developed in the broad concentration range (saliva 0.4-1000 ng/mL, urine 0.8-4000 ng/mL). The mean recoveries were 3.7 ± 7.6% for urine and 99.1 ± 2.6% for saliva. LOD for saliva was 0.12 ng/mL and for urine 0.05 ng/mL; LOQ was 0.4 ng/mL and 0.8 ng/mL, respectively. Intraday and interday precision equaled, respectively, 1.2% and 3.4% for urine, and 2.3% and 6.4% for saliva. There was a strong correlation between salivary cotinine and the uncorrected cotinine concentration in urine in the second and third trimesters of pregnancy. The cutoff values were established for saliva 12.9 ng/mL and urine 42.3 ng/mL or 53.1 μg/g creatinine with the ROC curve analysis. The developed analytical method was successfully applied to quantify cotinine, and a significant correlation between the urinary and salivary cotinine levels was found. The presented cut-off values for salivary and urinary cotinine ensure a categorization of the smoking status among pregnant women that is more accurate than self-reporting.

A pilot study on the feasibility of European harmonized Human Biomonitoring: Strategies towards a common approach, challenges and opportunities

In 2004 the European Commission and Member States initiated activities towards a harmonized approach for Human Biomonitoring surveys throughout Europe. The main objective was to sustain environmental health policy by building a coherent and sustainable framework and by increasing the comparability of data across countries. A pilot study to test common guidelines for setting up surveys was considered a key step in this process. Through a bottom-up approach that included all stakeholders, a joint study protocol was elaborated. From September 2011 till February 2012, 17 European countries collected data from 1844 mother-child pairs in the frame of DEMOnstration of a study to COordinate and Perform Human Biomonitoring on a European Scale (DEMOCOPHES).(1) Mercury in hair and urinary cadmium and cotinine were selected as biomarkers of exposure covered by sufficient analytical experience. Phthalate metabolites and Bisphenol A in urine were added to take into account increasing public and political awareness for emerging types of contaminants and to test less advanced markers/markers covered by less analytical experience. Extensive efforts towards chemo-analytical comparability were included. The pilot study showed that common approaches can be found in a context of considerable differences with respect to experience and expertize, socio-cultural background, economic situation and national priorities. It also evidenced that comparable Human Biomonitoring results can be obtained in such context. A European network was built, exchanging information, expertize and experiences, and providing training on all aspects of a survey. A key challenge was finding the right balance between a rigid structure allowing maximal comparability and a flexible approach increasing feasibility and capacity building. Next steps in European harmonization in Human Biomonitoring surveys include the establishment of a joint process for prioritization of substances to cover and biomarkers to develop, linking biomonitoring surveys with health examination surveys and with research, and coping with the diverse implementations of EU regulations and international guidelines with respect to ethics and privacy.

Exposure determinants of cadmium in European mothers and their children

The metal cadmium (Cd) is a widespread environmental pollutant with documented adverse effects on the kidneys and bones from long-term environmental exposure, but with insufficiently elucidated public health consequences such as risk of cardiovascular disease, hormone-related cancer in adults and developmental effects in children. This study is the first pan-European human biomonitoring project that succeeded in performing harmonized measurements of Cd in urine in a comparable way in mother-child couples from 16 European countries. The aim of the study was to evaluate the overall Cd exposure and significant determinants of Cd exposure. A study population of 1632 women (24-52 years of age), and 1689 children (5-12 years of age), from 32 rural and urban areas, was examined within a core period of 6 months in 2011-2012. Women were stratified as smokers and non-smokers. As expected, smoking mothers had higher geometric mean (gm) urinary cadmium (UCd; 0.24 µg/g crea; n=360) than non-smoking mothers (gm 0.18 µg/g crea; n=1272; p<0.0001), and children had lower UCd (gm 0.065 µg/g crea; n=1689) than their mothers at the country level. Non-smoking women exposed to environmental tobacco smoke (ETS) at home had 14% (95% CI 1-28%) higher UCd than those who were not exposed to ETS at home (p=0.04). No influence of ETS at home or other places on UCd levels was detected in children. Smoking women with primary education as the highest educational level of the household had 48% (95% CI 18-86%) higher UCd than those with tertiary education (p=0.0008). The same observation was seen in non-smoking women and in children; however they were not statistically significant. In children, living in a rural area was associated with 7% (95% CI 1-13%) higher UCd (p=0.03) compared to living in an urban area. Children, 9-12 years had 7% (95% CI 1-13%) higher UCd (p=0.04) than children 5-8 years. About 1% of the mothers, and 0.06% of the children, exceeded the tolerable weekly intake (TWI) appointed by EFSA, corresponding to 1.0 µg Cd/g crea in urine. Poland had the highest UCd in comparison between the 16 countries, while Denmark had the lowest. Whether the differences between countries are related to differences in the degree of environmental Cd contamination or to differences in lifestyle, socioeconomic status or dietary patterns is not clear.

Interpreting biomarker data from the COPHES/DEMOCOPHES twin projects: Using external exposure data to understand biomarker differences among countries

In 2011 and 2012, the COPHES/DEMOCOPHES twin projects performed the first ever harmonized human biomonitoring survey in 17 European countries. In more than 1800 mother-child pairs, individual lifestyle data were collected and cadmium, cotinine and certain phthalate metabolites were measured in urine. Total mercury was determined in hair samples. While the main goal of the COPHES/DEMOCOPHES twin projects was to develop and test harmonized protocols and procedures, the goal of the current paper is to investigate whether the observed differences in biomarker values among the countries implementing DEMOCOPHES can be interpreted using information from external databases on environmental quality and lifestyle. In general, 13 countries having implemented DEMOCOPHES provided high-quality data from external sources that were relevant for interpretation purposes. However, some data were not available for reporting or were not in line with predefined specifications. Therefore, only part of the external information could be included in the statistical analyses. Nonetheless, there was a highly significant correlation between national levels of fish consumption and mercury in hair, the strength of antismoking legislation was significantly related to urinary cotinine levels, and we were able to show indications that also urinary cadmium levels were associated with environmental quality and food quality. These results again show the potential of biomonitoring data to provide added value for (the evaluation of) evidence-informed policy making.