Phthalates and substitute plasticizers: Main achievements from the European human biomonitoring initiative HBM4EU.

Phthalates and the substitute plasticizer DINCH belong to the first group of priority substances investigated by the European Human Biomonitoring Initiative (HBM4EU) to answer policy-relevant questions and safeguard an efficient science-to-policy transfer of results. Human internal exposure levels were assessed using two data sets from all European regions and Israel. The first collated existing human biomonitoring (HBM) data (2005-2019). The second consisted of new data generated in the harmonized "HBM4EU Aligned Studies" (2014-2021) on children and teenagers for the ten most relevant phthalates and DINCH, accompanied by a quality assurance/quality control (QA/QC) program for 17 urinary exposure biomarkers. Exposures differed between countries, European regions, age groups and educational levels. Toxicologically derived Human biomonitoring guidance values (HBM-GVs) were exceeded in up to 5% of the participants of the HBM4EU Aligned Studies. A mixture risk assessment (MRA) including five reprotoxic phthalates (DEHP, DnBP, DiBP, BBzP, DiNP) revealed that for about 17% of the children and teenagers, health risks cannot be excluded. Concern about male reproductive health emphasized the need to include other anti-androgenic substances for MRA. Contaminated food and the use of personal care products were identified as relevant exposure determinants paving the way for new regulatory measures. Time trend analyses verified the efficacy of regulations: especially for the highly regulated phthalates exposure dropped significantly, while levels of the substitutes DINCH and DEHTP increased. The HBM4EU e-waste study, however, suggests that workers involved in e-waste management may be exposed to higher levels of restricted phthalates. Exposure-effect association studies indicated the relevance of a range of endpoints. A set of HBM indicators was derived to facilitate and accelerate science-to-policy transfer. Result indicators allow different groups and regions to be easily compared. Impact indicators allow health risks to be directly interpreted. The presented results enable successful science-to-policy transfer and support timely and targeted policy measures.

Compensated Hypogonadism Identified in Males with Cluster Headache: A Prospective Case-Controlled Study.

OBJECTIVE: Androgens have been hypothesized to be involved in the pathophysiology of cluster headache due to the male predominance, but whether androgens are altered in patients with cluster headache remains unclear. METHODS: We performed a prospective, case-controlled study in adult males with cluster headache. Sera were measured for hormones including testosterone, luteinizing hormone (LH), and sex hormone-binding globulin in 60 participants with episodic cluster headache (during a bout and in remission), 60 participants with chronic cluster headache, and 60 age- and sex-matched healthy controls. Free testosterone (fT) was calculated according to the Vermeulen equation. Shared genetic risk variants were assessed between cluster headache and testosterone concentrations. RESULTS: The mean fT/LH ratio was reduced by 35% (95% confidence interval [CI]: 21%-47%, p < 0.0001) in patients with chronic cluster headache and by 24% (95% CI: 9%-37%, p = 0.004) in patients with episodic cluster headache compared to controls after adjusting for age, sleep duration, and use of acute medication. Androgen concentrations did not differ between bouts and remissions. Furthermore, a shared genetic risk allele, rs112572874 (located in the intron of the microtubule associated protein tau (MAPT) gene on chromosome 17), between fT and cluster headache was identified. INTERPRETATION: Our results demonstrate that the male endocrine system is altered in patients with cluster headache to a state of compensated hypogonadism, and this is not an epiphenomenon associated with sleep or the use of acute medication. Together with the identified shared genetic risk allele, this may suggest a pathophysiological link between cluster headache and fT. ANN NEUROL 2024.

High incidence of imperforate vagina in ADGRA3-deficient mice.

BACKGROUND: Ten percent of the female population suffers from congenital abnormalities of the vagina, uterus, or oviducts, with severe consequences for reproductive and psychological health. Yet, the underlying causes of most of these malformations remain largely unknown. ADGRA3 (GPR125) is involved in WNT signaling and planar cell polarity, mechanisms vital to female reproductive tract development. Although ADGRA3 is a well-established spermatogonial stem cell marker, its role within the female urogenital system remains unclear. RESULTS: In this study, we found Adgra3 to be expressed throughout the murine female urogenital system, with higher expression pre-puberty than after sexual maturation. We generated a global Adgra3-/- mouse line and observed imperforate vagina in 44% of Adgra3-/- females, resulting in distension of the reproductive tract and infertility. Ovarian morphology, plasma estradiol, ovarian Cyp19a1, and vaginal estrogen receptor α (Esr1) expression were unaffected. However, compared to controls, a significantly lower bone mineral density was found in Adgra3-/- mice. Whereas vaginal opening in mice is an estrogen-dependent process, 17ß-estradiol treatment failed to induce vaginal canalization in Adgra3-/- mice. Furthermore, a marked reduction in vaginal and ovarian progesterone receptor expression was observed concomitant with an upregulation of apoptotic regulators Bcl2, Bid, and Bmf in adult Adgra3-/- females with a closed vagina. CONCLUSIONS: Our collective results shed new insights into the complex mechanisms by which the adhesion receptor ADGRA3 regulates distal vaginal tissue remodeling during vaginal canalization via altered sex hormone responsiveness and balance in apoptotic regulators. This highlights the potential of ADGRA3 as a target in diagnostic screening and/or therapy for obstructive vaginal malformations in humans.

Serum Phthalate Concentrations and Biomarkers of Oxidative Stress in Adipose Tissue in a Spanish Adult Cohort.

The relationship between phthalates, a group of chemical pollutants classified as endocrine disruptors, and oxidative stress is not fully understood. The aim of the present hospital-based study was to explore the associations between circulating levels of 10 phthalate metabolites and 8 biomarkers of oxidative stress in adipose tissue. The study population (n = 143) was recruited in two hospitals in the province of Granada (Spain). Phthalate metabolite concentrations were analyzed by isotope diluted online-TurboFlow-LC-MS/MS in serum samples, while oxidative stress markers were measured by commercially available kits in adipose tissue collected during routine surgery. Statistical analyses were performed by MM estimators' robust linear regression and weighted quantile sum regression. Mainly, positive associations were observed of monomethyl phthalate (MMP), monoiso-butyl phthalate (MiBP), and mono-n-butyl phthalate (MnBP) (all low molecular weight phthalates) with glutathione peroxidase (GPx) and thiobarbituric acid reactive substances (TBARS), while an inverse association was found between monoiso-nonyl phthalate (MiNP) (high molecular weight phthalate) and the same biomarkers. WQS analyses showed significant effects of the phthalate mixture on GSH (ß = -30.089; p-value = 0.025) and GSSG levels (ß = -19.591; p-value = 0.030). Despite the limitations inherent to the cross-sectional design, our novel study underlines the potential influence of phthalate exposure on redox homeostasis, which warrants confirmation in further research.

Cohort profile: The Copenhagen Analgesic Study-The COPANA cohort.

BACKGROUND: Development of the gonads during fetal life is complex and vital for adult reproductive health. Cell and animal studies have shown an alarming effect of mild analgesics on germ cells in both males and females. More than 50% of pregnant women use mild analgesics during pregnancy, which potentially could compromise the reproductive health of the next generation. OBJECTIVES: We present a research protocol designed to evaluate the effect of prenatal exposure to mild analgesics and endocrine-disrupting chemicals on gonadal function in the offspring. POPULATION: Healthy, singleton pregnant women and their partners. DESIGN: The COPANA cohort is a prospective, observational pregnancy and birth cohort. METHODS: Participants were enrolled during the first trimester of pregnancy. Information on the use of mild analgesics was collected retrospectively 3 months prior to pregnancy and prospectively every 2 weeks throughout the study. We collected extensive data on lifestyle and reproductive health. Biospecimens were collected in the first trimester (maternal and paternal urine- and blood samples), in the third trimester in conjunction with a study-specific ultrasound scan (maternal urine sample), and approximately 3 months post-partum during the infant minipuberty period (maternal and infant urine- and blood samples). A comprehensive evaluation of reproductive function in the infants during the minipuberty phase was performed, including an ultrasound scan of the testis or ovaries and uterus. PRELIMINARY RESULTS: In total, 685 pregnant women and their partners were included between March 2020 and January 2022. A total of 589 infants (287 males) and their parents completed the follow-up during the minipuberty phase (December 2020-November 2022). CONCLUSIONS: The Copenhagen Analgesic Study holds the potential to provide novel and comprehensive insights into the impact of early and late prenatal exposure to mild analgesics and other endocrine-disrupting chemicals on future reproductive function in the offspring.

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.

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.

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.

Pubertal timing, sex hormone levels, and associations between early life adversity and accelerated development amongst 11-year-old children of parents with schizophrenia or bipolar disorder and controls: The Danish high risk and Resilience study via 11.

Background: Children of parents with severe mental illness have several known risk factors for altered pubertal timing. Pubertal timing is important for children's physical and emotional development. We aimed to examine pubertal timing and associations between pubertal timing, early life adversity and child problem behavior including psychiatric diagnoses among children of parents with schizophrenia or bipolar disorder and controls. Methods: Self-reported Tanner stage (mean age 11.9, range 10.87-12.67), sex hormone levels, home environment, placement out of home, and problem behavior including psychiatric diagnoses of children at familial high-risk (FHR) of schizophrenia (FHR-SZ), bipolar disorder (FHR-BP) and population-based controls (PBC) were assessed. Results: A total of 465 children participated in the study (Tanner assessment N = 417, sex hormones N = 293). Assessed with self-reported Tanner, no difference in pubertal timing was found between groups (p = 0.09). Hormone levels did not differ between groups except for inhibin B (mean (SD) = 55.86 (29.13) pg/mL for FHR-SZ girls vs 84.98 (47.98) pg/mL) for PBC girls (p < 0.001)) and for follicle stimulating hormone (FSH) (mean (SD) = 5.82 (1.45) U/L for FHR-BP girls vs 4.54 (1.68) U/L for PBC girls (p < 0.001)). FHR children who were placed out of home (17 children, 3.8% of participants) had higher Tanner stages than those living at home (p < 0.001). Timing was not associated with level of problem behavior or psychiatric diagnoses. Conclusions: FHR children did not differ from controls in pubertal timing. Early life adversity assessed as placement out of home may be associated with accelerated pubertal timing among children of parents with schizophrenia or bipolar disorder.

Dexamethasone affects human fetal adrenal steroidogenesis and subsequent ACTH response in an ex vivo culture model.

Introduction: Administration of dexamethasone (DEX) has been used experimentally to suppress androgenization of external genitalia in 46,XX fetuses with congenital adrenal hyperplasia. Despite this, the prenatal biological mechanism-of-action of DEX on fetal development is not known. This study aimed to examine direct effects of DEX on human fetal adrenal (HFA) steroidogenic activity including possible effects on the subsequent response to ACTH-stimulation. Methods: Human fetal adrenal (HFA) tissue from 30 fetuses (1st trimester) were cultured ex vivo with A) DEX (10 µm) for 14 days, or B) DEX (10 µm) for 10 days followed by ACTH (1 nM) for 4 days. DEX-mediated effects on HFA morphology, viability, and apoptosis (immunohistochemistry), gene expression (quantitative PCR), and steroid hormone secretion (LC-MS/MS) were investigated. Results: DEX-treatment caused decreased androstenedione (p<0.05) and increased cortisol (p<0.01) secretion suggesting that direct effects on the adrenal gland may contribute to the negative feedback on the hypothalamic-pituitary-adrenal axis in vivo. An altered response to ACTH stimulation in HFA pre-treated with DEX included increased androgen (p<0.05) and reduced cortisol production (p<0.05), supporting clinical observations of a temporary decreased ACTH-response following prenatal DEX-treatment. Additionally, the secretion of corticosterone was decreased (p<0.0001) following ACTH-stimulation in the initially DEX-treated HFAs. Discussion: The observed effects suggest that prenatal DEX-treatment can cause direct effects on HFA steroidogenesis and in the subsequent response to ACTH-stimulation. This may indicate a requirement for careful monitoring of adrenal function in prenatally DEX-treated neonates, with particular focus on their mineralocorticoid levels.

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.

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.

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.

Maternal phthalate exposure during pregnancy and testis function of young adult sons.

BACKGROUND: Phthalate exposure during fetal life may disrupt testicular development. Congruent with this, studies have found shorter anogenital distance, reduced penile size and altered hormone levels in infant boys whose mothers were exposed to higher levels of some phthalates during pregnancy. Few studies have explored if such adverse effects persist in adulthood. Thus, we aimed to explore if there is an association between fetal phthalate exposure and markers of testicular function in young adult men. METHODS: In a longitudinal mother-child cohort from Copenhagen, Denmark, we examined 100 young men whose mothers during pregnancy had serum drawn and analyzed for 34 phthalate metabolites. Examinations of the young men took place at 18-20 years of age and included measurements of adult markers of testicular function (reproductive hormones, penile size, anogenital distance (AGD), testis volume, semen quality) and growth factors. Associations between maternal serum concentrations of phthalate metabolites and reproductive measures in the young men were tested using multiple linear regression. RESULTS: Most consistently, higher maternal phthalate exposure was associated with higher luteinizing hormone (LH) but unchanged testosterone in adult sons. Congruently, higher maternal exposure was associated with lower total and free testosterone/LH ratios in adult sons. For example, twice as high maternal MiNP was associated with a 7.9 % (95 % CI 1.6-13.8) lower free testosterone/LH ratio. There was no consistent pattern of associations between the different phthalate metabolites and other reproductive hormones, clinical outcomes, or semen quality. None of the tested associations was significant after multiplicity adjustment. CONCLUSIONS: In this exploratory study, higher maternal exposure to some phthalates was associated with impaired testicular Leydig cell function evidenced by a lower total and free testosterone/LH ratio in adult sons. This unique 18-20-year follow-up study raises concern and suggests that exposure of pregnant women to phthalates may have long-term effects on adult reproductive health in male offspring.

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.

Current exposure to phthalates and DINCH in European children and adolescents - Results from the HBM4EU Aligned Studies 2014 to 2021.

Phthalates are mainly used as plasticizers for polyvinyl chloride (PVC). Exposure to several phthalates is associated with different adverse effects most prominently on the development of reproductive functions. The HBM4EU Aligned Studies (2014-2021) have investigated current European exposure to ten phthalates (DEP, BBzP, DiBP, DnBP, DCHP, DnPeP, DEHP, DiNP, DiDP, DnOP) and the substitute DINCH to answer the open policy relevant questions which were defined by HBM4EU partner countries and EU institutions as the starting point of the programme. The exposure dataset includes ∼5,600 children (6-11 years) and adolescents (12-18 years) from up to 12 countries per age group and covering the North, East, South and West European regions. Study data from participating studies were harmonised with respect to sample size and selection of participants, selection of biomarkers, and quality and comparability of analytical results to provide a comparable perspective of European exposure. Phthalate and DINCH exposure were deduced from urinary excretions of metabolites, where concentrations were expressed as their key descriptor geometric mean (GM) and 95th percentile (P95). This study aims at reporting current exposure levels and differences in these between European studies and regions, as well as comparisons to human biomonitoring guidance values (HBM-GVs). GMs for children were highest for ∑DEHP metabolites (33.6 µg/L), MiBP (26.6 µg/L), and MEP (24.4 µg/L) and lowest for∑DiDP metabolites (1.91 µg/L) and ∑DINCH metabolites (3.57 µg/L). In adolescents highest GMs were found for MEP (43.3 µg/L), ∑DEHP metabolites (28.8 µg/L), and MiBP (25.6 µg/L) and lowest for ∑DiDP metabolites (= 2.02 µg/L) and ∑DINCH metabolites (2.51 µg/L). In addition, GMs and P95 stratified by European region, sex, household education level, and degree of urbanization are presented. Differences in average biomarker concentrations between sampling sites (data collections) ranged from factor 2 to 9. Compared to the European average, children in the sampling sites OCC (Denmark), InAirQ (Hungary), and SPECIMEn (The Netherlands) had the lowest concentrations across all metabolites and ESTEBAN (France), NAC II (Italy), and CROME (Greece) the highest. For adolescents, comparably higher metabolite concentrations were found in NEB II (Norway), PCB cohort (Slovakia), and ESTEBAN (France), and lower concentrations in POLAES (Poland), FLEHS IV (Belgium), and GerES V-sub (Germany). Multivariate analyses (Survey Generalized Linear Models) indicate compound-specific differences in average metabolite concentrations between the four European regions. Comparison of individual levels with HBM-GVs revealed highest rates of exceedances for DnBP and DiBP, with up to 3 and 5%, respectively, in children and adolescents. No exceedances were observed for DEP and DINCH. With our results we provide current, detailed, and comparable data on exposure to phthalates in children and - for the first time - in adolescents, and - for the first time - on DINCH in children and adolescents of all four regions of Europe which are particularly suited to inform exposure and risk assessment and answer open policy relevant questions.

Loss of Adgra3 causes obstructive azoospermia with high penetrance in male mice.

The adhesion receptor ADGRA3 (GPR125) is a known spermatogonial stem cell marker, but its impact on male reproduction and fertility has not been examined. Using a mouse model lacking Adgra3 (Adgra3-/- ), we show that 55% of the male mice are infertile from puberty despite having normal spermatogenesis and epididymal sperm count. Instead, male mice lacking Adgra3 exhibited decreased estrogen receptor alpha expression and transient dilation of the epididymis. Combined with an increased estradiol production, this indicates a post-pubertal hormonal imbalance and fluid retention. Dye injection revealed a blockage between the ejaculatory duct and the urethra, which is rare in mice suffering from infertility, thereby mimicking the etiologies of obstructive azoospermia found in human male infertility. To summarize, male reproductive tract development is dependent on ADGRA3 function that in concert with estrogen signaling may influence fluid handling during sperm maturation and storage.

Prenatal and current phthalate exposure and cognitive development in 7-year-old children from the Odense child cohort.

BACKGROUND: Phthalates are endocrine disrupters used in a variety of consumer products. Human studies suggest an association between phthalate exposure and cognitive development but adverse effects of the recently introduced phthalate substitutes have only been sparsely studied. OBJECTIVES: To investigate associations between prenatal and concurrent exposure to phthalates and IQ in 7-year-old children from the Odense Child Cohort. METHODS: Pregnant women from the Odense Child Cohort had phthalate metabolites measured in urine samples during 3rd trimester in 2010 to 2012. In addition, phthalates were also measured in urine samples from their offspring at age 7 years. IQ was assessed at age 7 years using four subtests from Wechsler Intelligence Scale for Children. The total study population consisted of 585 mother-child pairs with available prenatal urinary phthalate metabolites concentrations and IQ data at age 7 years. A subset of those (N = 274) had urinary phthalate metabolites measured in child urine at age 7 years. Phthalate concentrations were grouped into tertiles and associations with IQ were investigated using multiple linear regression adjusting for sex, maternal education and maternal/child BMI. RESULTS: Urinary phthalate metabolite concentrations both in pregnant women and children were generally lower compared to previous cohorts. Children with high prenatal urinary concentrations of MEP and metabolites of DEHP (∑DEHPm)(3rd tertile) had -3.1 (95% CI: -5.5, -0.6) (MEP) and - 3.0 (-5.5, -0.6) (∑DEHPm) IQ points at age 7 years compared to children with low concentrations (1st tertile). High concurrent urinary phthalate concentrations of MCPP, ∑DnHxPm, ∑DiDPm and ∑DiNPm in the 3rd tertile was associated with -3.7 (-7.2, -0.2), -4.4 (-7.9, -0.9), -3.7 (-7.2, -0.2) and - 5.6 (-9.1, -2.2) IQ points, respectively, compared to those with the lowest concentrations (1st tertile). CONCLUSION: We found significant inverse associations between some prenatal and concurrent urinary phthalate concentrations and IQ at age 7 years in this low exposed population. This suggests that exposure to phthalates both prenatally and during early childhood could be hazardous to child neurodevelopment, however, large-scale prospective studies assessing phthalate exposure through multiple urine samples, and possibly investigating cocktail effects of the chemicals as well as long term follow-up are warranted.

Urinary excretion of phthalates and the substitutes DINCH and DEHTP in Danish young men and German young adults between 2000 and 2017 - A time trend analysis.

Over the last twenty-five years it has become evident that exposure to several phthalates can have adverse effects on human health, such as endocrine disruption. This led to a series of EU regulations that resulted in a decrease in the production volumes of the restricted phthalates and an increased production of substitutes. The current study describes the impact of regulations and changes in production and use of phthalates and their substitutes on internal exposure patterns in two European populations since the beginning of the 2000'ies. Using harmonised data from young adults in Denmark (Danish Young Men Study, n = 1,063, spot urine) and Germany (Environmental Specimen Bank, n = 878, 24-h urine) with repeated cross-sectional design (3-11 cycles per biomarker) we applied Locally Estimated Scatterplot Smoothing (LOESS) and Generalized Linear Models (GLMs) to estimate time trends and the role of covariates on the trend (e.g. age, BMI). Time trends of daily excretion (µg/24h) are comparable between the two samples for the regulated (DEHP, BBzP, DiNP, DnBP, DiBP, DiDP/DPHP) as well as the non-regulated substances (DMP, DEP, DINCH, DEHTP) although the rate of change differ for some of the compounds. GLM results indicate that the daily excretion of the most regulated phthalates has decreased over time (DEHP yearly about 12-16%, BBzP 5%, DnBP 0.3-17%, and DiBP 4-12%). Interestingly, also the non-regulated phthalates DMP and DEP decreased by 6-18% per year. In sharp contrast, the phthalate substitutes DINCH and DEHTP show very steep annual increases (∼10-68% and ∼100%, respectively) between 2009 and 2017. We did not find an effect of age, sex, BMI, or education on the time trend. The present study provides comparable insights into how exposure to phthalates and two of their substitutes have changed over the last two decades in Germany and Denmark.

Prenatal paraben exposures and birth size: Sex-specific associations in a healthy population - A study from the Odense Child Cohort.

OBJECTIVES: To investigate the sex-specific associations between maternal paraben concentrations in second trimester urine and birth size of the offspring. METHODS: A prospective cohort study of 529 mother-child pairs within the Odense Child Cohort. Pregnant women were recruited to the cohort from 2010 to 2012 and provided fasting spot urine samples in second trimester (median 28.7 weeks). Concentrations of methylparaben (MeP), ethylparaben (EtP), iso-propylparaben (i-PrP), n-propylparaben (n-PrP), n-butylparaben (n-BuP) and benzylparaben (BzP) were analyzed by isotope diluted liquid-chromatography tandem-mass-spectrometry and osmolality adjusted. Exposures were categorized into tertiles or above/below level of detection. Data on maternal and birth characteristics were extracted from hospital records. Sex-stratified multiple linear regression analyses were performed according to relevant birth outcomes (length, weight, head/abdominal circumference) adjusting for a priori defined confounders. RESULTS: Higher paraben levels were detected in pregnant women who were older, more obese, who smoked and were primigravidae. Generally, higher maternal paraben exposure was consistently associated with lower birth size in female but not in male offspring, but with few substantial or statistically significant. Higher maternal exposure to n-BuP during pregnancy was associated with a statistically significant lower birth size in female offspring only [birth weight: -137 g (95 % CI -256; -19), head circumference: -0.48 cm (95 % CI -0.90; -0.05), abdominal circumference: -0.65 cm (95 % CI -1.21; -0.08)]. No differences in birth size were observed for other parabens. CONCLUSION: Higher maternal exposure to n-butylparaben was associated with lower birth size in female but not male offspring.