BACKGROUND:Exposure to endocrine-disrupting chemicals (EDCs) during fetal life is associated with reproductive disorders in humans. While research has largely focused on environmental EDCs and developmental exposure, some pharmaceuticals, including commonly used antifungal agents, also possess endocrine-disrupting properties. We investigate whether prenatal exposure to over-the-counter antifungal drugs is associated with altered fetal and postnatal steroidogenesis and reproductive development. METHODS:We combined epidemiological, experimental, and translational approaches using data from a prospective mother-child cohort (n = 589), ex vivo human fetal adrenal cultures, and in vivo rat models. Maternal antifungal use during pregnancy was assessed prospectively, and associations with fetal growth, anogenital distance (AGD), penile measurements, and circulating steroid hormone levels during infancy were evaluated. Experimental studies examined the effects of selected antifungal agents on steroidogenesis in cultured human fetal adrenals and gestationally exposed Sprague-Dawley rats. RESULTS:Early-pregnancy antifungal exposure is associated with reduced circulating steroid hormone levels and indicators of diminished androgen action, including decreased penile width at infancy. In contrast, exposure during mid-to-late pregnancy is associated with elevated steroid hormone levels and longer AGD in both female fetuses and male infants. Consistent with these observations, the antifungal agent clotrimazole reduces progesterone levels in human fetal adrenal cultures and in gestationally exposed rats, whereas miconazole increases AGD in rats. CONCLUSIONS:These translational findings suggest that commonly used antifungal agents alter fetal adrenal steroidogenesis and influence reproductive development, with effects persisting into postnatal life. Given their widespread use and over-the-counter availability, the safety of topical azole use during pregnancy may warrant re-evaluation.

STUDY QUESTION:Is fetal exposure to paracetamol associated with markers of ovarian function in infancy? SUMMARY ANSWER:Mild to moderate doses of prenatal paracetamol exposure, assessed by detailed maternal reports and urinary measurements, are associated with ovarian morphology and activity as well as reduced size of estrogen-responsive tissues in infant girls. WHAT IS KNOWN ALREADY:Maternal use of paracetamol is widespread. Across multiple independent animal studies, fetal exposure consistently impairs the formation of primordial ovarian follicles, causing subfertility and premature estropause in female offspring. STUDY DESIGN SIZE DURATION:The Copenhagen Analgesic study (COPANA) is a single-center, prospective, observational cohort study conducted at a university hospital (March 2020 to November 2022). From 3425 eligible participants, 685 healthy singleton pregnant women of Caucasian origin, were enrolled in the first trimester of pregnancy, and 302 infant daughters were examined at follow-up. Exclusion criteria included maternal diabetes or thyroid disease, pre- or post-term delivery, or severe infant illness. PARTICIPANTS/MATERIALS SETTING METHODS:Pregnant women reported paracetamol use biweekly and provided first-trimester urinary samples which were analyzed for paracetamol levels (LC-MS/MS) and adjusted for urinary osmolarity (n = 299). Girls were classified by timing of initial exposure: early fetal life (<17 weeks, n = 92), mid-late fetal life (≥17 weeks, n = 67), or unexposed controls (n = 143). Of the 92 girls initially exposed in early fetal life, a subgroup was exposed exclusively in early fetal life (n = 22).In an independent confirmatory cohort, 1210 girls were followed from infancy to adolescence. Their exposure was maternal self-reported (any) use of paracetamol during pregnancy and reported early in the third trimester (yes/no). MAIN RESULTS AND ROLE OF CHANCE:Early fetal exposure was associated with reduced ovarian volume (-0.11 cm3, 95% CI -0.19 to -0.03) and reduced uterine volume (-0.18 cm3, -0.35 to -0.01), whereas mid-late fetal exposure was associated with fewer ovarian follicles (-1.05 follicles, -1.71 to -0.39) compared to unexposed girls. AMH levels were lower in girls exposed exclusively in early fetal life (-0.45 SDS, -0.87 to -0.03) compared to unexposed girls. Maternal urinary paracetamol concentrations were inversely associated with ovarian and uterine volume as well as breast tissue diameter. In the independent confirmatory cohort, fetal paracetamol exposure was associated with reduced uterine volume at puberty (-4.11 cm3, -7.29 to -0.92) and smaller ovarian volume during adolescence (-2.76 cm3, -4.82 to -0.70). LIMITATIONS REASON FOR CAUTION:The observational design of the study allows evaluation of exposure outcome associations, while the causality interpretation is supported by consistency with experimental models demonstrating comparable effects. Residual confounding by indication for paracetamol use cannot be completely excluded, although the results were robust after accounting for fever and other maternal factors. The analytic design of the current study limited our ability to evaluate whether frequency or patterns of paracetamol use influenced the observed associations. WIDER IMPLICATIONS OF THE FINDINGS:In this prospective cohort study of infant girls, prenatal exposure to paracetamol was associated with differences in several markers of ovarian development, including ovarian volume, follicle number, and circulating AMH, as well as uterine volume and breast tissue diameter. The associations with reduced ovarian and uterine size were also observed in a confirmatory cohort followed to adolescence. Whether these early-life differences have long-term clinical relevance, including effects on reproductive lifespan, remains uncertain and requires analyses in studies with extended follow-up. FUNDING:This research was supported by Rigshospitalets Research Council under grant (E-22717-21), Læge Sofus Carl Emil Friis og hustru Doris Friis'Legat (F-23936-01), Aase og Ejnar Danielsens Foundation (20-10-0367), Helsefonden (20-B-0388), Axel Muusfeldt Foundation (2020-0385), and The Danish Centre for Endocrine Disrupting Substances (CeHoS) (2022-23219). The funders had no role in considering the study design or in the collection, analysis, interpretation of data, writing of the report, or decision to submit the article for publication. The publication was completed as part of the MERLON project under grant agreement No. 101137411. The MERLON project is funded by the European Union. Views and opinions expressed are, however, those of the authors only and do not necessarily reflect those of the European Union or the European Health and Digital Executive Agency (HADEA). Neither the European Union nor HADEA can be held responsible for them. DISCLOSURES:All authors declare no support from any organization for the submitted work, no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years, and no other relationships or activities that could appear to have influenced the submitted work. One author (MA) is a government official. TRIAL REGISTRATION NUMBER:ClinicalTrials.gov ID: NCT0436922.

Recent decades have seen declining total fertility rates (TFR) globally, alongside increased use of assisted reproductive technology (ART). However, TFR includes ART births and excludes induced abortions, complicating assessments of population fecundity. Here, we examine trends in cohort total fertility rates (cTFR), induced abortions, and ART use through a nationwide cohort study of 1,648,971 pregnancies, including abortions, among Danish women aged 15-45 years born between 1958 and 1999. A new index, the Comprehensive Unassisted Pregnancy Rate (live births and induced abortions, excluding ART births), was developed. Our findings reveal a decline in unassisted pregnancy rates for women born after 1961, with an accelerated decline for those born after 1970. In contrast, cTFR increased for women born between 1958 and 1970 before decreasing, driven by trends in induced abortions and ART births. These differences highlight a disconnect between cTFR and fecundity measures. The declining unassisted pregnancy rates, reduced abortions, and increased ART demand raise concerns about population fecundity. Understanding these trends' biological and socioeconomic drivers requires large-scale, transdisciplinary studies of representative populations. Our results emphasize the need for alternative measures, like the Comprehensive Unassisted Pregnancy Rate, to assess reproductive health and fertility trends accurately.

Fertility rates have fallen to unprecedentedly low levels worldwide, reflecting a widespread fertility crisis. Although this decline has often been attributed to social and economic shifts, growing evidence suggests that biological factors may also play a role. Markers of decreased fecundity are on the rise, with an increasing number of couples requiring medically assisted reproduction because of poor semen quality and/or female infertility. In addition, the number of children born after assisted reproduction is rising. Trends in pregnancies terminated by an induced abortion may also provide important information regarding fecundity. A new index, the comprehensive unassisted pregnancy rate (defined by live births and induced abortions, excluding births as a result of artificial reproductive techniques), has been developed to reflect the fecundity of a population. Recent data from a few high-income countries have shown that the comprehensive unassisted pregnancy rate has been steadily declining across successive decades. If this finding is replicated in other countries, it could indicate that patterns of changing fecundity may be emerging as a population-wide international phenomenon.

Objective: To describe the bioactivity of circulating androgens during pubertal transition as determined by an in vitro bioassay assessing androgen receptor (AR) activation, and to compare these findings with circulating concentrations of total testosterone (total T) measured by LC-MS/MS. Methods: This longitudinal study included ten healthy boys from the Copenhagen Puberty Study II conducted from 2006 to 2011. Main Outcome Measure(S)Androgen bioactivity and serum concentrations of total T were measured by an in vitro bioassay and LC-MS/MS with limits of detection of 0.3 and 0.01 nmol/L, respectively. The serum concentration of free testosterone (free T) was calculated using the Vermeulen equation. Pubertal onset was defined as testicular enlargement >= 4 mL, assessed by palpation. Results: Androgen bioactivity was unmeasurable before puberty but measurable in nine of ten boys 0.8-1.7 years after pubertal onset. Total T and free T were measurable prepubertally in all boys and increased in two before pubertal onset. Androgen bioactivity correlated strongly with total T (r = 0.93, p < 0.001) and free T (r = 0.93, p < 0.001). ROC accuracies were 77% for androgen bioactivity, 93% for total T, and 95% for free T. Conclusion: Androgen bioactivity was undetectable before pubertal onset but measurable after in most boys, reflecting both the bioassay's lower sensitivity and potential physiological changes in androgen bioactivity during early puberty. Further studies are needed to clarify these observations.

BACKGROUND:Endocrine science remains underrepresented in European Union research programs despite the fundamental role of hormone health in human wellbeing. Analysis of the CORDIS database reveals a persistent gap between the societal impact of endocrine disorders and their research prioritization. At national funding level, endocrine societies report limited or little attention of national research funding towards endocrinology. The EndoCompass project-a joint initiative between the European Society of Endocrinology and the European Society of Paediatric Endocrinology, aimed to identify and promote strategic research priorities in endocrine science to address critical hormone-related health challenges. METHODS:Research priorities were established through comprehensive analysis of the EU CORDIS database covering the Horizon 2020 framework period (2014-2020). Expert consultation was conducted to identify key research priorities, followed by broader stakeholder engagement including society members and patient advocacy groups. RESULTS:Research priorities encompass variations in sex development, hypothalamic-pituitary-gonadal regulation, and female and male reproductive disorders. Key areas include improving diagnostic capacity through (epi)genetic analysis, optimizing hormonal treatments, developing fertility preservation strategies. Special emphasis is placed on establishing pan-European registries, developing novel reproductive technologies, and exploring environmental impacts on reproductive health. CONCLUSIONS:This component of the EndoCompass project provides an evidence-based roadmap for strategic research investment. This framework identifies crucial investigation areas into reproductive and developmental endocrinology pathophysiology, prevention, and treatment strategies, ultimately aimed at reducing the burden of these disorders on individuals and society. The findings support the broader EndoCompass objective of aligning research funding with areas of the highest potential impact in endocrine health.

BACKGROUND:Di-n-hexyl phthalate (DnHxP) is one of the most potent phthalates with adverse effects on the male reproductive system. Despite a European ban on DnHxP since 2020, high urinary excretion of mono-n-hexyl phthalate (MnHxP), the major metabolite of DnHxP, has been observed in recent European human biomonitoring (HBM) studies. Sunscreen products containing the UV-filter diethylamino hydroxybenzyl hexyl benzoate (DHHB), which can be contaminated with DnHxP, were pointed out as a relevant source of exposure to DnHxP. OBJECTIVE:To study possible seasonal variation in urinary excretion of MnHxP in different Danish study populations. METHOD:MnHxP was measured by LC-MS/MS in 1591 urine samples collected in 2016-2022 from pregnant women, 7-year-old children, and infants and their parents participating in three different cohort studies. RESULTS:Urinary MnHxP was above the limit of detection (0.04 μg/L) in 84 % and above 1 μg/L in 33 % of samples. The 7-year-old children was the study population with the highest excretion rate (97 %). The highest urinary MnHxP concentration (72.5 μg/L) exceeded the health-based guidance value (HBM-I value) of 60 μg/L. A significantly higher urinary excretion of MnHxP was observed in the summer compared to the winter season in all study populations. 80 % of the urine samples with MnHxP concentrations > 1 μg/L were from the summer season. 40 % of Danish sunscreen products contain DHHB. CONCLUSION:The marked seasonal variation with higher MnHxP excretions in the summer season supports the hypothesis that the recently unveiled DnHxP contamination of the UV-filter DHHB in sunscreen products could be driving these exposures.

Vaginal candidiasis affects about 20 % of pregnant women and is usually treated with over-the-counter topical antifungal medication (azoles). Vaginal or transdermal application of azoles are absorbed and detectable in circulation. Azoles inhibit CYP51, which is crucial for the integrity of fungal cellular membranes. However, cell cultures have shown that azoles also affect steroidogenesis. This study investigated maternal antifungal application during pregnancy and the association with reproductive hormones during minipuberty and anogenital distance (AGD) the in the offspring from infancy to 9 years of age. In the Odense Child Cohort (2010-2012), women completed questionnaires about antifungal application during pregnancy. Serum concentrations of luteinising hormone (LH), follicle-stimulating hormone (FSH), testosterone (T), estrone (E1), estradiol (E2), Δ4-androstenedione (adione), 17α-hydroxyprogesterone (17-OHP) and dehydroepiandrosterone-sulphate (DHEAS) were analysed in 454 infants at 3 months. AGD was assessed at 3, 18 months and 3, 5, 7 and 9 years of age, with 1792 measurements. Topical antifungal application during pregnancy was reported by 35 women. In boys, maternal application before GW 19 was associated with shorter AGD as well as lower adrenal hormone levels. In girls, application before GW 19 was associated with longer AGD and lower reproductive and adrenal hormone levels, while application after GW 19 was associated with shorter AGD, while hormone levels did not differ. Given the small number of cases, the findings should be interpreted with caution. The widespread use of over-the-counter antifungals appears to affect AGD and hormone production in offspring, which is concerning and may have long-term consequences for reproductive health.

Polycyclic aromatic hydrocarbons (PAHs) are persistent environmental pollutants with well-documented associations to adverse health effects, posing significant public health challenges across Europe. Human exposure to 13 urinary PAH metabolites was assessed in a harmonized cohort of European adults aged 20-39, representing diverse geographic regions across Europe: North (Iceland and Denmark), East (Poland and the Czech Republic), South (Croatia and Portugal), and West (France, Germany, Switzerland, and Luxembourg). This study aimed to achieve a unified understanding of PAH exposure by employing stringent participant selection criteria and harmonizing biomarker analyses by utilizing high-quality analytical protocols across multiple laboratories in Europe. Key findings revealed consistently elevated metabolite levels in smokers compared to non-smokers, with naphthalene metabolites dominating the profiles over phenanthrene and fluorene derivatives. Country-specific analyses highlighted Poland as having the highest naphthalene metabolite concentrations, while Luxembourg exhibited elevated pyrene metabolite levels. Urbanization influenced exposure, with slightly higher metabolite concentrations in town populations compared to rural areas. While sex-based stratification revealed no marked differences, gender emerged as a significant covariate in regression models, with women generally displaying higher exposure to naphthalene metabolites. Educational level further stratified exposure, with lower education correlating with increased PAH levels. Multivariate linear regression identified key exposure factors, including sampling season (i.e., summer, winter, autumn, and spring), dietary habits e.g., smoked foods, and proximity to smoke-prone environments. This dataset provides a significant baseline for evaluating the European Commission's Chemicals Strategy for Sustainability (CSS) and underscores the utility of harmonized human biomonitoring studies in informing targeted public health interventions.

BACKGROUND:Endocrine science remains underrepresented in European Union research programmes despite the fundamental role of hormone health in human well-being. Analysis of the CORDIS database reveals a persistent gap between the societal impact of endocrine disorders and their research prioritization. At national funding level, endocrine societies report limited or little attention of national research funding towards endocrinology. The EndoCompass project-a joint initiative between the European Society of Endocrinology and the European Society of Paediatric Endocrinology, aimed to identify and promote strategic research priorities in endocrine science to address critical hormone-related health challenges. METHODS:Research priorities were established through a comprehensive analysis of the EU CORDIS database covering the Horizon 2020 framework period (2014-2020). An expert analysis was conducted by leading researchers in environmental endocrinology, integrating literature review, epidemiological evidence, and emerging research priorities to identify key challenges and opportunities across endocrine systems. RESULTS:Research priorities span 5 critical domains: mechanisms and biomarkers of endocrine-disrupting chemical (EDC) actions; environmental pharmaceutical contamination; climate change effects on endocrine function; endocrine consequences of air and water pollution; and mechanisms linking environmental stress to hormone disruption. Special emphasis is placed on understanding developmental programming, transgenerational effects, and implications for public health policy. CONCLUSIONS:This component of the EndoCompass project provides an evidence-based roadmap for strategic research investment. The analysis demonstrates that environmental factors like EDCs fundamentally impact multiple endocrine systems, requiring coordinated research approaches. The findings support the broader EndoCompass objective of aligning research funding with areas of highest potential impact in endocrine health.

Bisphenol A (BPA) is widely used in the manufacturing of plastics. BPA can pass the placental barrier and influence fetal development. Due to its estrogenic and anti-androgenic properties, BPA may contribute sex-specific differences in developmental effects. We examined associations between maternal urinary concentrations of BPA and birth size. In this cohort study of 832 mother-child pairs from the Odense Child Cohort, pregnant women provided spot urine samples at gestational week 28, which were analyzed for BPA by isotope diluted LC-MS/MS. Osmolality adjusted urinary BPA concentrations were categorized into quartiles. Mother-child characteristics were obtained from hospital records and questionnaires. Linear regression analyses examining the association between BPA concentrations and offspring birth size (weight, length, head, and abdominal circumference) were performed for the full cohort and stratified by offspring sex. BPA was detected above the limit of detection in 85

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.

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.

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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.