Prenatal phthalate exposure and pubertal development in 16-year-old daughters: reproductive hormones and number of ovarian follicles.

STUDY QUESTION: Is there a possible association between prenatal phthalate exposure and late effects in teenage daughters with respect to reproductive hormone levels, uterine volume, and number of ovarian follicles? SUMMARY ANSWER: Our study showed subtle associations between phthalate metabolite concentrations in maternal serum from pregnancy or cord blood and LH and insulin-like growth factor 1 (IGF-1) levels as well as uterine volume in their daughters 16 years later. WHAT IS KNOWN ALREADY: Endocrine-disrupting environmental chemicals may adversely affect human reproductive health, and many societies have experienced a trend toward earlier puberty and an increasing prevalence of infertility in young couples. The scientific evidence of adverse effects of foetal exposure to a large range of chemicals, including phthalates, on male reproductive health is growing, but very few studies have explored effects on female reproduction. STUDY DESIGN, SIZE, DURATION: This follow-up study included 317 teenage daughters who were part of the Copenhagen Mother-Child Cohort, a population-based longitudinal birth cohort of 1210 females born between 1997 and 2002. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 317 female participants (median age 16 years) were examined for weight, height, and menstrual pattern. A serum sample was analysed for concentrations of reproductive hormones, and trans-abdominal 3D ultrasonography was performed to obtain the number of ovarian follicles, ovarian and uterine size. Prenatal maternal serum samples were available for 115 females, and cord blood samples were available for 118 females. These were analysed for concentrations of 32 phthalate metabolites. Weighted quantile sum regression was used for modelling associations of combined prenatal phthalate exposure with the reproductive outcomes in post-menarcheal females. MAIN RESULTS AND THE ROLE OF CHANCE: In bivariate correlation analyses, negative significant associations were found between several prenatal phthalate metabolite concentrations and serum hormone concentrations (testosterone, 17-OH-progesterone, and IGF-1) as well as number of ovarian follicles in puberty. Positive significant correlations were found between prenatal phthalate exposure and FSH and sex hormone-binding globulin concentrations. Combined analyses of phthalate exposure (weighted quantile sums) showed significant negative associations with IGF-1 concentration and uterine volume as well as a significant positive association with LH concentration. LIMITATIONS, REASONS FOR CAUTION: Phthalate metabolites were measured in serum from single prenatal maternal blood samples and cord blood samples. Potential concomitant exposure to other endocrine-disrupting environmental chemicals before or after birth was not controlled for. The study population size was limited. WIDER IMPLICATIONS OF THE FINDINGS: Our results support the need for further research into possible adverse effects of environmental chemicals during foetal development of the female reproductive system. STUDY FUNDING/COMPETING INTEREST(S): The work was supported by The Center on Endocrine Disruptors (CeHoS) under The Danish Environmental Protection Agency and The Ministry of Environment and Food (grant number: MST-621-00 065). No conflicts of interest are declared. TRIAL REGISTRATION NUMBER: N/A.

Exposure to bisphenol A in European women from 2007 to 2014 using human biomonitoring data - The European Joint Programme HBM4EU.

BACKGROUND: Bisphenol A (BPA; or 4,4'-isopropylidenediphenol) is an endocrine disrupting chemical. It was widely used in a variety of plastic-based manufactured products for several years. The European Food Safety Authority (EFSA) recently reduced the Tolerable Daily Intake (TDI) for BPA by 20,000 times due to concerns about immune-toxicity. OBJECTIVE: We used human biomonitoring (HBM) data to investigate the general level of BPA exposure from 2007 to 2014 of European women aged 18-73 years (n = 4,226) and its determinants. METHODS: Fifteen studies from 12 countries (Austria, Belgium, Denmark, France, Germany, Greece, Israel, Luxembourg, Slovenia, Spain, Sweden, and the United Kingdom) were included in the BPA Study protocol developed within the European Joint Programme HBM4EU. Seventy variables related to the BPA exposure were collected through a rigorous post-harmonization process. Linear mixed regression models were used to investigate the determinants of total urine BPA in the combined population. RESULTS: Total BPA was quantified in 85-100 % of women in 14 out of 15 contributing studies. Only the Austrian PBAT study (Western Europe), which had a limit of quantification 2.5 to 25-fold higher than the other studies (LOQ=2.5 µg/L), found total BPA in less than 5 % of the urine samples analyzed. The geometric mean (GM) of total urine BPA ranged from 0.77 to 2.47 µg/L among the contributing studies. The lowest GM of total BPA was observed in France (Western Europe) from the ELFE subset (GM=0.77 µg/L (0.98 µg/g creatinine), n = 1741), and the highest levels were found in Belgium (Western Europe) and Greece (Southern Europe), from DEMOCOPHES (GM=2.47 µg/L (2.26 µg/g creatinine), n = 129) and HELIX-RHEA (GM=2.47 µg/L (2.44 µg/g creatinine), n = 194) subsets, respectively. One hundred percent of women in 14 out of 15 data collections in this study exceeded the health-based human biomonitoring guidance value for the general population (HBM-GVGenPop) of 0.0115 µg total BPA/L urine derived from the updated EFSA's BPA TDI. Variables related to the measurement of total urine BPA and those related to the main socio-demographic characteristics (age, height, weight, education, smoking status) were collected in almost all studies, while several variables related to BPA exposure factors were not gathered in most of the original studies (consumption of beverages contained in plastic bottles, consumption of canned food or beverages, consumption of food in contact with plastic packaging, use of plastic film or plastic containers for food, having a plastic floor covering in the house, use of thermal paper…). No clear determinants of total urine BPA concentrations among European women were found. A broader range of data planned for collection in the original questionnaires of the contributing studies would have resulted in a more thorough investigation of the determinants of BPA exposure in European women. CONCLUSION: This study highlights the urgent need for action to further reduce exposure to BPA to protect the population, as is already the case in the European Union. The study also underscores the importance of pre-harmonizing HBM design and data for producing comparable data and interpretable results at a European-wide level, and to increase HBM uptake by regulatory agencies.

Sex- and age-specific reference intervals of 16 steroid metabolites quantified simultaneously by LC-MS/MS in sera from 2458 healthy subjects aged 0 to 77 years.

BACKGROUND: Reference intervals covering the whole life span for all the metabolites in the steroid hormone biosynthesis quantified by sensitive and robust analytical methods are sparse or not existing. OBJECTIVE: To develop a state-of-the-art LC-MS/MS method for simultaneous quantification of multiple steroid metabolites and to establish detailed sex- and age-specific reference intervals for 16 steroid metabolites. MATERIALS AND METHOD: An isotope diluted LC-MS/MS method was developed for simultaneous quantitation of 16 steroid hormones. Serum samples from cross-sectional cohorts of healthy infants, children, adolescents, and adults aged 0.17 months to 77 years (n = 2458) were analysed. RESULTS: With this novel, specific, and sensitive LC-MS/MS method, it was possible to quantify progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone sulfate, androstenedione, testosterone, dihydrotestosterone, 11-deoxycorticosterone, corticosterone, 11-deoxycortisol, cortisol, and cortisone in ≥90 % of the samples, while estrone sulfate, aldosterone and dehydroepiandrosterone were quantified in 77 %, 75 % and 60 % of the samples, respectively. 21-deoxycortisol was only detectable in 2.5 % of samples from healthy subjects. Sex- and age-dependent fluctuations observed in minipuberty, puberty and adulthood including the menopausal transition were modelled. This enabled us to establish valid reference intervals from birth to late adult life for both males and females. CONCLUSION: Detailed sex- and age-specific reference intervals of multiple, simultaneously quantified steroid metabolites by a novel and specific LC-MS/MS method provides a valuable tool for clinical practice and for future research.

Injection of luteinizing hormone or human chorionic gonadotropin increases calcium excretion and serum PTH in males.

Animal and human studies have suggested that sex steroids have calciotropic actions, and it has been proposed that follicle-stimulating hormone (FSH) may exert direct effects on bone. Here, we demonstrate the expression of the receptor for Luteinizing hormone (LH) and human choriogonadotropin (hCG), LHCGR, in human kidney tissue, suggesting a potential influence on calcium homeostasis. To investigate the role of LHCGR agonist on calcium homeostasis in vivo, we conducted studies in male mice and human subjects. Male mice were treated with luteinizing hormone (LH), and human extrapolation was achieved by injecting 5000 IU hCG once to healthy men or men with hypergonadotropic or hypogonadotropic hypogonadism. In mice, LH treatment significantly increased urinary calcium excretion and induced a secondary increase in serum parathyroid hormone (PTH). Similarly, hCG treatment in healthy men led to a significant increase in urinary calcium excretion, serum PTH levels, and 1,25 (OH)2D3, while calcitonin, and albumin levels were reduced, possibly to avoid development of persistent hypocalcemia. Still, the rapid initial decline in ionized calcium coincided with a significant prolongation of the cardiac QTc-interval that normalized over time. The observed effects may be attributed to LH/hCG-receptor (LHCGR) activation, considering the presence of LHCGR expression in human kidney tissue, and the increase in sex steroids occurred several hours after the changes in calcium homeostasis. Our translational study shed light on the intricate relationship between gonadotropins, sex hormones and calcium, suggesting that LHCGR may be influencing calcium homeostasis directly or indirectly.

Serum Concentrations of Inhibin B in Healthy Females and Males Throughout Life.

OBJECTIVE: To describe the natural history of inhibin B throughout life according to sex, age, and pubertal development. METHODS: Based on serum samples from 2707 healthy controls aged 0 to 80 years, sex- and age-specific reference ranges of inhibin B concentrations were constructed. Concentrations were evaluated according to pubertal development and use of oral contraceptives (OCs). Also, measurements from 42 patients with Klinefelter syndrome were included. RESULTS: In both sexes, inhibin B concentrations were high during minipuberty, decreased in childhood, and increased significantly from Tanner stages B1 to B3 (peak: B4) in females and from G1 to G3 (peak: G3) in males. Despite variations in menstruating females, inhibin B concentrations remained relatively constant after puberty, until becoming unmeasurable at menopause. Despite a modest decrease, the inhibin B concentration in males remained relatively high from puberty onwards. At any age, males had highest concentrations. Inhibin B standard deviation (SD) scores were lower in OC-users (median SD score = -0.88) than in non-users (SD score = 0.35), p < 0.001. In patients with Klinefelter syndrome, inhibin B concentrations spanned the reference range until around 15 years of age, where they decreased to subnormal or unmeasurable levels. CONCLUSION: Valuable sex- and age-specific reference data for inhibin B concentrations were provided. In OC-users, decreased concentrations of inhibin B underlined the ovaries as the only place of inhibin B production. In patients with Klinefelter syndrome, the decline in inhibin B concentrations at puberty underlined the shift in regulation of inhibin B production at pubertal onset.

Prenatal and childhood exposure to bisphenols and bone mineral density in 7-year-old children from the Odense Child Cohort.

BACKGROUND: Bisphenol A (BPA) is a well-known endocrine disrupter used in several consumer products. Restricted use of BPA has led to increased use of bisphenol F (BPF) and bisphenol S (BPS). While previous studies found no associations between prenatal BPA and BPF exposure and bone mineral density (BMD), two recent cohort studies found that prenatal BPS exposure was negatively associated with bone mineral density in the offspring. AIM: To determine possible associations between maternal and child urinary bisphenol concentrations, BMD and bone mineral content (BMC) in 7-year-old healthy children. METHODS: Pregnant women were recruited in 2010-2012 to participate in the Odense Child Cohort (OCC), Denmark. Maternal urine samples were collected in gestational week 28 and urinary BPA concentration was measured by isotope diluted LC-MS/MS. The children delivered a urine sample at age 7 years in which BPA, BPF and BPS were measured by an extended LS-MS/MS method based on the original method. At age 7 years DXA scans were performed and BMC and Z-score for BMD calculated. Associations between osmolality adjusted urinary maternal BPA and child BPA, BPF and BPS concentrations and BMC and BMD Z-score were examined by multiple linear regression analysis adjusted for potential confounders. Additionally, a combined effect of the bisphenols were evaluated by including the sum of child urinary BPA, BPF and BPS concentrations in the statistical analyses. RESULTS: A total of 546 mothers and 453 children aged 7 years participated. BPA was detected in 84% and 96% of the maternal and child urine samples, respectively. We found no significant association between maternal urinary BPA concentration during pregnancy and BMC and BMD Z-score in 7-year-old children. In addition, no association between current bisphenol exposure in tertiles and bone density was found, interestingly, current BPA and summed bisphenol exposure in the highest 10% was associated with lower BMD Z-score at age 7-years, statistically significant for boys. CONCLUSION: In these low exposed children we found no association between prenatal or current bisphenol exposure in tertiles and BMD in healthy children, however, the highest 10% exposed children had lower BMD, significant for boys, suggesting a negative impact with high bisphenol exposure. The short half-lives of bisphenols and the cross-sectional nature of the child exposure prompt more longitudinal studies to further clarify this topic.

Enhanced identification of endocrine disruptors through integration of science-based regulatory practices and innovative methodologies: The MERLON Project.

The prevalence of hormone-related health issues caused by exposure to endocrine disrupting chemicals (EDCs) is a significant, and increasing, societal challenge. Declining fertility rates together with rising incidence rates of reproductive disorders and other endocrine-related diseases underscores the urgency in taking more action. Addressing the growing threat of EDCs in our environment demands robust and reliable test methods to assess a broad variety of endpoints relevant for endocrine disruption. EDCs also require effective regulatory frameworks, especially as the current move towards greater reliance on non-animal methods in chemical testing puts to test the current paradigm for EDC identification, which requires that an adverse effect is observed in an intact organism. Although great advances have been made in the field of predictive toxicology, disruption to the endocrine system and subsequent adverse health effects may prove particularly difficult to predict without traditional animal models. The MERLON project seeks to expedite progress by integrating multispecies molecular research, new approach methodologies (NAMs), human clinical epidemiology, and systems biology to furnish mechanistic insights and explore ways forward for NAM-based identification of EDCs. The focus is on sexual development and function, from foetal sex differentiation of the reproductive system through mini-puberty and puberty to sexual maturity. The project aims are geared towards closing existing knowledge gaps in understanding the effects of EDCs on human health to ultimately support effective regulation of EDCs in the European Union and beyond.

Longitudinal evaluation of fetal and infant AGD in healthy children: association with penile size, testosterone and DHT.

CONTEXT: The anogenital distance (AGD) is considered a postnatal readout of early fetal androgen action. Little is known of prenatal AGD and how it correlates with AGD postnatally. OBJECTIVES: We present longitudinal measurements of fetal- and infant AGD. We evaluate the impact of testosterone and dihydrotestosterone at minipuberty on AGD and penile size. DESIGN: Secondary analyses of an observational, prospective pregnancy and birth cohort, COPANA (2020-2022). SETTING: Copenhagen University Hospital - Rigshospitalet. PARTICIPANTS: 685 healthy, singleton pregnant women enrolled, 657 women attended 3rd trimester ultrasound, 589 infants completed follow-up. MAIN OUTCOME MEASURES: 3rd trimester ultrasound (GW29-34): Fetal AGD. Minipuberty clinical examination (app. 3.5 months postpartum): infant AGD, penile width and stretched length (SPL), circulating testosterone and dihydrotestosterone (LC-MS/MS). RESULTS: AGD was available in 650/657 fetuses (310 boys) and 588/589 infants (287 boys). Boys had longer fetal and infant AGD compared to girls; fetal AGDas: mean (SD) 21.4 mm (±3.5), fetal AGDaf: 12.8 mm (±2.3), p < 0.001, infant AGDas: 32.0 mm (±5.6) and infant AGDaf: 15.8 (±3.3), p < 0.001. Fetal AGD correlated with infant AGD in boys and girls (Spearman's r = 0.275, p < 0.001 and r = 0.189, p = 0.001 respectively), but not with circulating testosterone or dihydrotestosterone at minipuberty. Penile size correlated positively with circulating androgen levels at minipuberty, i.e.: SPL vs testosterone: r = 0.235, p < 0.001. CONCLUSIONS: AGD is sexual dimorphic already in the 3rd trimester. Fetal and infant AGD correlates. AGD is associated with body size but not circulating androgen levels at minipuberty. These findings suggest that fetal and infant AGD, reflect androgen action during early fetal development.

The epidemiology of cryptorchidism and potential risk factors, including endocrine disrupting chemicals.

Congenital cryptorchidism, also known as undescended testis, is the condition where one or both testes are not in place in the scrotum at birth and is one of the most common birth defects in boys. Temporal trends and geographic variation in the prevalence of cryptorchidism from 1% to 9% have been reported in prospective cohort studies. The testes develop in the abdominal cavity and descend to the scrotum in two phases, which should be completed by gestational week 35. Thus, the risk of cryptorchidism is higher in preterm boys. In many cases a spontaneous descent occurs during the first months of life during the surge of gonadotropins and testosterone. If not, the testis is usually brought down to the scrotum, typically by surgery, to increase future fertility chances and facilitate cancer surveillance. The increasing frequency of impaired semen quality and testicular cancer, with which cryptorchidism is associated, represents a concern for male reproductive health in general and a need to understand its risk factors. The risk of cryptorchidism is closely related to gestational factors (preterm birth, low birth weight and intrauterine growth restriction), and especially maternal smoking seems to be a risk factor. Evidence is accumulating that the increasing prevalence of cryptorchidism is also related to prenatal exposure to environmental chemicals, including endocrine disrupting compounds. This association has been corroborated in rodents and supported by ecological studies. Conducting human studies to assess the effect of endocrine disrupting chemicals and their interactions is, however, challenged by the widespread concomitant exposure of all humans to a wide range of chemicals, the combined effect of which and their interactions are highly complex.

Antibiotic Use in Dental Care of Dogs, Cats, and Rabbits in Sweden.

Antimicrobial resistance is one of the largest threats to global health. In society as well as in healthcare facilities, antimicrobial resistance is rapidly increasing with the main reason being overuse and misuse of antibiotics combined with inadequate infection prevention. For humans, dental care accounts for about 10% of all antibiotic prescriptions, making it an important target for antibiotic stewardship interventions. Corresponding figures for veterinary care are currently lacking but dental disease is frequently diagnosed in small animals. An important first step in the work towards prudent use of antibiotics is to understand antibiotic prescription habits and through that estimate the adherence to veterinary antibiotic guidelines as well as the need for education, training, and improved policies. The aim of this article is to present the results of a multicentre point prevalence survey sent to Swedish IVC Evidensia practices during autumn 2021 to recognize the use of antibiotics associated with dental treatments in dogs, cats, and rabbits. During the study period, 4.4% of the dental patients in Swedish IVC Evidensia small animal veterinary practices received antibiotics. The most used antibiotics prescribed were ampicillin, amoxicillin, and clindamycin indicating an overall high level of compliance to veterinary dental guidelines. This article demonstrates that Swedish veterinarians use antibiotics prudently in small animal dentistry and the results may be used as a future global benchmark.

Ovarian follicular fluid levels of phthalates and benzophenones in relation to fertility outcomes.

BACKGROUND: Many endocrine disrupting chemicals (EDCs), for instance phthalates and benzophenones, are associated with adverse fertility outcomes and semen quality parameters. OBJECTIVE: To evaluate if concentrations of selected phthalate metabolites and benzophenones measured in follicular fluid are associated with fertility outcomes (i.e., reproductive hormones, antral follicle count, detected heartbeat at gestational week 7, and live birth) and, in a supplementary study, if measured concentrations of chemicals in follicular fluid can exert biological effects on human spermatozoa. METHODS: Overall, 111 couples from a fertility clinic in Denmark contributed with 155 follicular fluid samples. Concentrations of 43 metabolites from 19 phthalates and phthalate substitutes and six benzophenones were measured in follicular fluid using liquid chromatography-tandem mass spectrometry. Multiple linear and logistic regression with an applied generalized estimating equation model allowing more than one measurement per woman assessed the association between follicular EDC levels and fertility outcomes. The assessment of biological effects of individual and mixtures of EDCs on human spermatozoa was conducted through a human sperm cell based Ca2+-fluorimetric assay. RESULTS: Benzophenone-3 (BP-3) and seven metabolites of five phthalates were detectable in follicular fluid. Women with metabolites of dibutyl phthalate isomers in the highest tertiles had lower antral follicle count (MiBP: ß = -5.35 [95 % CI: -9.06; -2.00], MnBP: ß = -5.25 [95 % CI: -9.00; -2.00]) and lower odds for detecting a heartbeat at gestational week 7 (MiBP: OR = 0.35 [95 % CI: 0.14; 0.91], MnBP: OR = 0.39 [95 % CI: 0.13; 1.15]). Mixtures of the measured concentrations of BP-3 and the seven phthalate metabolites induced a small significant increase in the intracellular calcium ion concentration in human spermatozoa from healthy donors (n = 3). DISCUSSION: Phthalate metabolites and BP-3 were detectable in follicular fluid and high concentrations of some phthalate metabolites were linked with lower chance of successful fertility treatment outcomes. Chemical mixture concentrations in follicular fluid induced a calcium response in human spermatozoa highlighting possible biological effects at physiologically relevant concentrations.

Evaporation of serum after long-term biobank storage: A chemical analysis of maternal serum from a large Danish pregnancy screening registry.

BACKGROUND: Relying on freezer stored biospecimens is preferred in epidemiolocal studies exploring environmental pregnancy exposures and later offspring health. Storage duration may increase the pre-analytical variability, potentially adding measurement uncertainty. We investigated evaporation of maternal serum after long-term biobank storage using ions (sodium, Na+; chloride, Cl-) recognized for stability and relatively narrow normal biological reference ranges in human serum. METHODS: A chemical analysis study of 275 biobanked second trimester maternal serum from a large Danish pregnancy screening registry. Serum samples were collected between 1985-1995 and stored at -20°C. Ion concentrations were quantified with indirect potentiometry using a Roche Cobas 6000 analyzer and compared according to storage time and normal biological ranges in second trimester. Ion concentrations were also compared with normal biological variation assessed by baseline Na+ and Cl- serum concentrations from a separate cohort of 24,199 non-pregnant women measured before freezing with the same instrument. RESULTS: The overall mean ion concentrations in biobanked serum were 147.5 mmol/L for Na+ and 109.7 for Cl-. No marked linear storage effects were observed according to storage time. Ion concentrations were consistently high across sampling years, especially for specific sampling years, and a relatively large proportion were outside respective normal ranges in second trimester: 38.9% for Na+ and 43.6% for Cl-. Some variation in concentrations was also evident in baseline serum used as quality controls. CONCLUSIONS: Elevated ion concentrations suggest evaporation, but independent of storage duration in the present study (27-37 years). Any evaporation may have occurred prior to freezer storage or during the first 27 years. Other pre-analytical factors such as low serum volume have likely influenced the concentrations, particularly given the high within year variability. Overall, we consider the biobanked serum samples internally comparable to enable their use in epidemiological studies.

Time Patterns in Internal Human Exposure Data to Bisphenols, Phthalates, DINCH, Organophosphate Flame Retardants, Cadmium and Polyaromatic Hydrocarbons in Europe.

Human biomonitoring (HBM) data in Europe are often fragmented and collected in different EU countries and sampling periods. Exposure levels for children and adult women in Europe were evaluated over time. For the period 2000-2010, literature and aggregated data were collected in a harmonized way across studies. Between 2011-2012, biobanked samples from the DEMOCOPHES project were used. For 2014-2021, HBM data were generated within the HBM4EU Aligned Studies. Time patterns on internal exposure were evaluated visually and statistically using the 50th and 90th percentiles (P50/P90) for phthalates/DINCH and organophosphorus flame retardants (OPFRs) in children (5-12 years), and cadmium, bisphenols and polycyclic aromatic hydrocarbons (PAHs) in women (24-52 years). Restricted phthalate metabolites show decreasing patterns for children. Phthalate substitute, DINCH, shows a non-significant increasing pattern. For OPFRs, no trends were statistically significant. For women, BPA shows a clear decreasing pattern, while substitutes BPF and BPS show an increasing pattern coinciding with the BPA restrictions introduced. No clear patterns are observed for PAHs or cadmium. Although the causal relations were not studied as such, exposure levels to chemicals restricted at EU level visually decreased, while the levels for some of their substitutes increased. The results support policy efficacy monitoring and the policy-supportive role played by HBM.

Maternal concentrations of phthalates and Attention-Deficit Hyperactivity Disorder (ADHD-) related symptoms in children aged 2 to 4 years from Odense child cohort.

BACKGROUND: Phthalates are endocrine disrupting chemicals used in everyday consumer products. Several epidemiological studies have examined the association between prenatal phthalate concentration and Attention-Deficit Hyperactivity Disorder (ADHD) in offspring, but the findings have been inconclusive. OBJECTIVES: To investigate the association between maternal urinary concentrations of phthalate metabolites during pregnancy and ADHD related symptoms in children at 2 to 4 years in a large prospective cohort. METHODS: In the Odense Child Cohort from Denmark were women recruited in early pregnancy from 2010 to 2012. Phthalate concentrations were measured in urine samples collected in 3rd trimester and separated into low and high weight phthalates. Parents filled in the Child Behavior Checklist for ages 1.5 to 5 years (CBCL/1½-5), including a 6-item ADHD symptom scale at children aged 2 to 4 years. Data were analysed by use of adjusted negative binomial regression. RESULTS: A total of 658 mother-child pairs were included. Urinary phthalate metabolite concentrations were generally low compared to previous cohorts. A doubling in maternal concentration of the low-weighted phthalate metabolite MCPP was significantly associated with lower ADHD symptoms score in children (IRR: 0.95 (95 % CI 0.91-0.98)), strongest in girls (IRR: 0.92 (0.87-0.98)). Sex differences were observed. High maternal phthalate metabolite concentrations were associated with lower ADHD symptom score in girls, significant trends across tertile of MCPP and MnBP (p = 0.018, p = 0.038, respectively). In boys, maternal concentrations of high-molecular-weight phthalates (MBzP, ∑DiNP and ∑DEHP) were associated with an almost significantly higher ADHD symptom score (IRR for a doubling in concentration: 1.04 (95 % CI: 0.99-1.10), IRR: 1.05 (95 % CI: 0.97-1.13), IRR: 1.04 (95 % CI: 0.99-1.10), respectively). CONCLUSION: Maternal concentration of the low-weighted phthalate metabolite MCPP was significantly associated with a lower ADHD symptom score in children, strongest in girls. Maternal concentrations of high-molecular-weight phthalates were associated with non-significant increase in ADHD symptom score in boys.

A vision for safer food contact materials: Public health concerns as drivers for improved testing.

Food contact materials (FCMs) and food contact articles are ubiquitous in today's globalized food system. Chemicals migrate from FCMs into foodstuffs, so called food contact chemicals (FCCs), but current regulatory requirements do not sufficiently protect public health from hazardous FCCs because only individual substances used to make FCMs are tested and mostly only for genotoxicity while endocrine disruption and other hazard properties are disregarded. Indeed, FCMs are a known source of a wide range of hazardous chemicals, and they likely contribute to highly prevalent non-communicable diseases. FCMs can also include non-intentionally added substances (NIAS), which often are unknown and therefore not subject to risk assessment. To address these important shortcomings, we outline how the safety of FCMs may be improved by (1) testing the overall migrate, including (unknown) NIAS, of finished food contact articles, and (2) expanding toxicological testing beyond genotoxicity to multiple endpoints associated with non-communicable diseases relevant to human health. To identify mechanistic endpoints for testing, we group chronic health outcomes associated with chemical exposure into Six Clusters of Disease (SCOD) and we propose that finished food contact articles should be tested for their impacts on these SCOD. Research should focus on developing robust, relevant, and sensitive in-vitro assays based on mechanistic information linked to the SCOD, e.g., through Adverse Outcome Pathways (AOPs) or Key Characteristics of Toxicants. Implementing this vision will improve prevention of chronic diseases that are associated with hazardous chemical exposures, including from FCMs.

Urinary concentration of phthalates and bisphenol A during minipuberty is associated with reproductive hormone concentrations in infant boys.

BACKGROUND: The transient postnatal activation of the hypothalamic-pituitary-gonadal hormone axis is termed minipuberty and considered an important developmental period, which is highly sensitive to endocrine disruption. Here, we explore exposure-outcome associations during minipuberty between concentrations of potentially endocrine disrupting chemicals (EDCs) in urine of infant boys and their serum reproductive hormone concentrations. METHODS: In total, 36 boys participating in the COPENHAGEN Minipuberty Study had data available for both urine biomarkers of target endocrine disrupting chemicals and reproductive hormones in serum from samples collected on the same day. Serum concentrations of reproductive hormones were measured by immunoassays or by LC-MS/MS. Urinary concentrations of metabolites of 39 non-persisting chemicals, including phthalates and phenolic compounds, were measured by LC-MS/MS. Nineteen chemicals had concentrations above the limit of detection in ≥50% of children and were included in data analysis. Associations of urinary phthalate metabolite and phenol concentrations (in tertiles) with hormone outcomes (age- and sex-specific SD-scores) were analysed by linear regression. Primarily, we focused on the EU regulated phthalates; butylbenzyl phthalate (BBzP), di-iso-butyl phthalate (DiBP), di-n-butyl phthalate (DnBP), and di-(2-ethylhexyl) phthalate (DEHP) as well as bisphenol A (BPA). Urinary metabolites of DiBP, DnBP and DEHP were summed and expressed as ∑DiBPm, ∑DnBPm and ∑DEHPm. RESULTS: Compared to boys in the lowest ∑DnBPm tertile, urinary concentration of ∑DnBPm was associated with concurrent higher luteinizing hormone (LH) and anti-Müllerian hormone (AMH) SD-scores as well as lower testosterone/LH ratio in boys in the middle ∑DnBPm tertile (estimates (CI 95%) 0.79 (0.04; 1.54), 0.91 (0.13; 1.68), and -0.88 (-1.58;-0.19), respectively). Further, higher insulin-like peptide 3 (INSL3) SD-scores and lower DHEAS SD-score in boys in the highest ∑DnBPm tertile (0.91 (0.12; 1.70) and -0.85 (-1.51;-0.18), respectively) were observed. In addition, boys in the middle and highest ∑DEHPm tertile had higher LH (1.07 (0.35; 1.79) and 0.71 (-0.01; 1.43), respectively) and in the highest ∑DEHPm tertile also higher AMH (0.85 (0.10; 1.61)) concentration SD-scores, respectively. Boys in the highest BPA tertile had significantly higher AMH and lower DHEAS concentration compared to boys in the lowest BPA tertile (1.28 (0.54; 2.02) and -0.73 (-1.45; -0.01)), respectively. DISCUSSION: Our findings indicate that exposure to chemicals with known or suspected endocrine disrupting potential, especially the EU-regulated DnBP, DEHP and BPA, may modify male reproductive hormone concentrations in infant boys suggesting that minipuberty is a critical window sensitive to endocrine disruption.

Low serum anti-Müllerian hormone is associated with semen quality in infertile men and not influenced by vitamin D supplementation.

BACKGROUND: Anti-Müllerian hormone (AMH) is released by testicular Sertoli cells and of great importance during fetal male sexual development, but less is known about the role of circulating AMH during adulthood. In vitro studies have shown that vitamin D may induce AMH transcription, but a controlled trial investigating the possible effect of vitamin D on serum AMH has not been conducted in men. METHODS: A single-center, double-blinded, randomized placebo-controlled clinical trial (NCT01304927) conducted in Copenhagen, Denmark. A total of 307 infertile men were included and randomly assigned (1:1) to a single dose of 300,000 IU cholecalciferol followed by 1400 IU cholecalciferol + 500 mg of calcium daily (n = 151) or placebo (n = 156) for 150 days. Difference in serum AMH was a predefined secondary endpoint. Explorative outcomes were associations between serum AMH and gonadal function in infertile men. The primary endpoint was difference in semen quality and has previously been published. RESULTS: Infertile men in the lowest AMH tertile had significantly lower sperm concentration (∆T3-1 16 mill/mL (228%); P < 0.001), sperm count (∆T3-1 55 million (262%); P < 0.001), motile sperm count (∆T3-1 28 million (255%); P < 0.001), progressive motile sperm count (∆T3-1 18 million (300%); P < 0.001), testis size (∆T3-1 2.7 mL (16%); P < 0.001), serum inhibin B (∆T3-1 72 pg/mL (59%); P < 0.001), inhibin B/FSH ratio (∆T3-1 48 (145%); P < 0.001), and higher FSH (∆T3-1 2.6 (38%); P < 0.001) than the tertile of infertile men with highest serum AMH. Vitamin D supplementation had no effect on serum AMH compared with placebo treatment. CONCLUSIONS: In infertile men, low serum AMH is associated with severely impaired gonadal function illustrated by poor semen quality and lower testosterone/LH ratio. Serum AMH in infertile men was not influenced by vitamin D supplementation.

Exposure to Phthalates in European Children, Adolescents and Adults since 2005: A Harmonized Approach Based on Existing HBM Data in the HBM4EU Initiative.

Phthalates are mainly used as plasticizers and are associated inter alia with adverse effects on reproductive functions. While more and more national programs in Europe have started monitoring internal exposure to phthalates and its substitute 1,2-Cyclohexanedicarboxylic acid (DINCH), the comparability of results from such existing human biomonitoring (HBM) studies across Europe is challenging. They differ widely in time periods, study samples, degree of geographical coverage, design, analytical methodology, biomarker selection, and analytical quality assurance level. The HBM4EU initiative has gathered existing HBM data of 29 studies from participating countries, covering all European regions and Israel. The data were prepared and aggregated by a harmonized procedure with the aim to describe-as comparably as possible-the EU-wide general population's internal exposure to phthalates from the years 2005 to 2019. Most data were available from Northern (up to 6 studies and up to 13 time points), Western (11; 19), and Eastern Europe (9; 12), e.g., allowing for the investigation of time patterns. While the bandwidth of exposure was generally similar, we still observed regional differences for Butyl benzyl phthalate (BBzP), Di(2-ethylhexyl) phthalate (DEHP), Di-isononyl phthalate (DiNP), and Di-isobutyl phthalate (DiBP) with pronounced decreases over time in Northern and Western Europe, and to a lesser degree in Eastern Europe. Differences between age groups were visible for Di-n-butyl phthalate (DnBP), where children (3 to 5-year olds and 6 to 11-year olds) had lower urinary concentrations than adolescents (12 to 19-year-olds), who in turn had lower urinary concentrations than adults (20 to 39-year-olds). This study is a step towards making internal exposures to phthalates comparable across countries, although standardized data were not available, targeting European data sets harmonized with respect to data formatting and calculation of aggregated data (such as developed within HBM4EU), and highlights further suggestions for improved harmonization in future studies.

Benzophenone-3: Comprehensive review of the toxicological and human evidence with meta-analysis of human biomonitoring studies.

BACKGROUND: Benzophenone-3 (BP-3) and its major metabolite benzophenone-1 (BP-1) are widely used as UV filters in sunscreens and cosmetics to prevent sunburn and skin damage, or as stabilizers to prevent photodegradation in many commercial products. As a result, their presence is ubiquitous in the environment, wildlife and humans. Based on endocrine disruption concerns, international regulatory agencies are performing a closer evaluation. OBJECTIVE AND METHODS: This work aimed to comprehensively review the available human relevant evidence for safety issues in MEDLINE/PubMed in order to create a structured database of studies, as well as to conduct an integrative analysis as part of the Human Biomonitoring for Europe (HBM4EU) Initiative. RESULTS: A total of 1,635 titles and abstracts were screened and 254 references were evaluated and tabulated in detail, and classified in different categories: i) exposure sources and predictors; ii) human biomonitoring (HBM) exposure levels to perform a meta-analysis; iii) toxicokinetic data in both experimental animals and humans; iv) in vitro and in vivo rodent toxicity studies; and v) human data on effect biomarkers and health outcomes. Our integrative analysis showed that internal peak BP-3 concentrations achieved after a single whole-body application of a commercially available sunscreen (4% w/w) may overlap with concentrations eliciting endocrine disrupting effects in vitro, and with internal concentrations causing in vivo adverse female reproductive effects in rodents that were supported by still limited human data. The adverse effects in rodents included prolonged estrous cycle, altered uterine estrogen receptor gene expression, endometrium hyperplasia and altered proliferation and histology of the mammary gland, while human data indicated menstrual cycle hormonal alterations and increased risk of uterine fibroids and endometriosis. Among the modes of action reported (estrogenic, anti-androgenic, thyroid, etc.), BP-3 and especially BP-1 showed estrogenic activity at human-relevant concentrations, in agreement with the observed alterations in female reproductive endpoints. The meta-analysis of HBM studies identified a higher concern for North Americans, showing urinary BP-3 concentrations on average 10 and 20 times higher than European and Asian populations, respectively. DISCUSSION AND CONCLUSIONS: Our work supports that these benzophenones present endocrine disrupting properties, endorsing recent European regulatory efforts to limit human exposure. The reproducible and comprehensive database generated may constitute a point of departure in future risk assessments to support regulatory initiatives. Meanwhile, individuals should not refrain from sunscreen use. Commercially available formulations using inorganic UV filters that are practically not absorbed into systemic circulation may be recommended to susceptible populations.

Harmonized human biomonitoring in European children, teenagers and adults: EU-wide exposure data of 11 chemical substance groups from the HBM4EU Aligned Studies (2014-2021).

As one of the core elements of the European Human Biomonitoring Initiative (HBM4EU) a human biomonitoring (HBM) survey was conducted in 23 countries to generate EU-wide comparable HBM data. This survey has built on existing HBM capacity in Europe by aligning national or regional HBM studies, referred to as the HBM4EU Aligned Studies. The HBM4EU Aligned Studies included a total of 10,795 participants of three age groups: (i) 3,576 children aged 6-12 years, (ii) 3,117 teenagers aged 12-18 years and (iii) 4,102 young adults aged 20-39 years. The participants were recruited between 2014 and 2021 in 11-12 countries per age group, geographically distributed across Europe. Depending on the age group, internal exposure to phthalates and the substitute DINCH, halogenated and organophosphorus flame retardants, per- and polyfluoroalkyl substances (PFASs), cadmium, bisphenols, polycyclic aromatic hydrocarbons (PAHs), arsenic species, acrylamide, mycotoxins (deoxynivalenol (total DON)), benzophenones and selected pesticides was assessed by measuring substance specific biomarkers subjected to stringent quality control programs for chemical analysis. For substance groups analyzed in different age groups higher average exposure levels were observed in the youngest age group, i.e., phthalates/DINCH in children versus teenagers, acrylamide and pesticides in children versus adults, benzophenones in teenagers versus adults. Many biomarkers in teenagers and adults varied significantly according to educational attainment, with higher exposure levels of bisphenols, phthalates, benzophenones, PAHs and acrylamide in participants (from households) with lower educational attainment, while teenagers from households with higher educational attainment have higher exposure levels for PFASs and arsenic. In children, a social gradient was only observed for the non-specific pyrethroid metabolite 3-PBA and di-isodecyl phthalate (DiDP), with higher levels in children from households with higher educational attainment. Geographical variations were seen for all exposure biomarkers. For 15 biomarkers, the available health-based HBM guidance values were exceeded with highest exceedance rates for toxicologically relevant arsenic in teenagers (40%), 3-PBA in children (36%), and between 11 and 14% for total DON, Σ (PFOA + PFNA + PFHxS + PFOS), bisphenol S and cadmium. The infrastructure and harmonized approach succeeded in obtaining comparable European wide internal exposure data for a prioritized set of 11 chemical groups. These data serve as a reference for comparison at the global level, provide a baseline to compare the efficacy of the European Commission's chemical strategy for sustainability and will give leverage to national policy makers for the implementation of targeted measures.