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.

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.

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.

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.

Cadmium exposure in adults across Europe: Results from the HBM4EU Aligned Studies survey 2014-2020.

The objectives of the study were to estimate the current exposure to cadmium (Cd) in Europe, potential differences between the countries and geographic regions, determinants of exposure and to derive European exposure levels. The basis for this work was provided by the European Human Biomonitoring Initiative (HBM4EU) which established a framework for alignment of national or regional HBM studies. For the purpose of Cd exposure assessment, studies from 9 European countries (Iceland, Denmark, Poland, Czech Republic, Croatia, Portugal, Germany, France, Luxembourg) were included and urine of 20-39 years old adults sampled in the years 2014-2021 (n = 2510). The measurements in urine were quality assured by the HBM4EU quality assurance/quality control scheme, study participants' questionnaire data were post-harmonized. Spatially resolved external data, namely Cd concentrations in soil, agricultural areas, phosphate fertilizer application, traffic density and point source Cd release were collected for the respective statistical territorial unit (NUTS). There were no distinct geographic patterns observed in Cd levels in urine, although the data revealed some differences between the specific study sites. The levels of exposure were otherwise similar between two time periods within the last decade (DEMOCOPHES - 2011-2012 vs. HBM4EU Aligned Studies, 2014-2020). The age-dependent alert values for Cd in urine were exceeded by 16% of the study participants. Exceedances in the different studies and locations ranged from 1.4% up to 42%. The studies with largest extent of exceedance were from France and Poland. Association analysis with individual food consumption data available from participants' questionnaires showed an important contribution of vegetarian diet to the overall exposure, with 35% higher levels in vegetarians as opposed to non-vegetarians. For comparison, increase in Cd levels due to smoking was 25%. Using NUTS2-level external data, positive associations between HBM data and percentage of cropland and consumption of Cd-containing mineral phosphate fertilizer were revealed, which indicates a significant contribution of mineral phosphate fertilizers to human Cd exposure through diet. In addition to diet, traffic and point source release were identified as significant sources of exposure in the study population. The findings of the study support the recommendation by EFSA to reduce Cd exposure as also the estimated mean dietary exposure of adults in the EU is close or slightly exceeding the tolerable weekly intake. It also indicates that regulations are not protecting the population sufficiently.

In vitro investigation of endocrine disrupting effects of pesticides on Ca2+-signaling in human sperm cells through actions on the sperm-specific and steroid-activated CatSper Ca2+-channel.

BACKGROUND: Ca2+-signaling controls sperm cell functions necessary for successful fertilization. Multiple endocrine disrupting chemicals have been found to interfere with normal Ca2+-signaling in human sperm cells through an activation of the sperm-specific CatSper Ca2+-channel, which is vital for normal male fertility. OBJECTIVES: We investigated 53 pesticides for their ability to interfere with CatSper mediated Ca2+-signaling and function in human sperm cells. METHODS: Effects of the pesticides on Ca2+-signaling in human sperm cells were evaluated using a Ca2+-fluorometric assay. Effects via CatSper were assessed using the specific CatSper inhibitor RU1968. Effects on human sperm function and viability were assessed using an image cytometry-based acrosome reaction assay and the modified Kremer's sperm-mucus penetration assay. RESULTS: 28 of 53 pesticides were found to induce Ca2+-signals in human sperm cells at 10 µM. The majority of these 28 active pesticides induced Ca2+-signals through CatSper and interfered with subsequent Ca2+-signals induced by the two endogenous CatSper ligands progesterone and prostaglandin E1. Multiple active pesticides were found to affect Ca2+-mediated sperm functions and viability at 10 µM. Low nM dose mixtures of the active pesticides alone or in combination with other environmental chemicals were found to significantly induce Ca2+-signals and inhibit Ca2+-signals induced subsequently by progesterone and prostaglandin E1. CONCLUSIONS: Our results show that pesticides, both alone and in low nM dose mixtures, interfere with normal Ca2+-signaling in human sperm cells in vitro in low nM concentrations. Biomonitoring of the active pesticides in relevant matrices such as blood and reproductive fluids is very limited and the effects of real time human pesticide exposure on human sperm cells and fertility thus remains largely unknown. To which extent human pesticide exposure affects the chances of a successful fertilization in humans in vivo needs further research.

Environmental factors in declining human fertility.

A severe decline in child births has occurred over the past half century, which will lead to considerable population declines, particularly in industrialized regions. A crucial question is whether this decline can be explained by economic and behavioural factors alone, as suggested by demographic reports, or to what degree biological factors are also involved. Here, we discuss data suggesting that human reproductive health is deteriorating in industrialized regions. Widespread infertility and the need for assisted reproduction due to poor semen quality and/or oocyte failure are now major health issues. Other indicators of declining reproductive health include a worldwide increasing incidence in testicular cancer among young men and alterations in twinning frequency. There is also evidence of a parallel decline in rates of legal abortions, revealing a deterioration in total conception rates. Subtle alterations in fertility rates were already visible around 1900, and most industrialized regions now have rates below levels required to sustain their populations. We hypothesize that these reproductive health problems are partially linked to increasing human exposures to chemicals originating directly or indirectly from fossil fuels. If the current infertility epidemic is indeed linked to such exposures, decisive regulatory action underpinned by unconventional, interdisciplinary research collaborations will be needed to reverse the trends.

Assessment of biomarkers in pediatric atopic dermatitis by tape strips and skin biopsies.

BACKGROUND: The cytokine profile of atopic dermatitis (AD) depends on age, ethnicity, and disease severity. This study examined biomarkers in children with AD collected by tape strips and skin biopsies, and examined whether the levels differed with filaggrin genotype, disease severity, and food allergy. METHODS: Twenty-five children aged 2-14 years with AD were clinically examined. Skin biopsies were collected from lesional skin and tape strips were collected from lesional and non-lesional skin. We analyzed natural moisturizing factor (NMF) and 17 immune markers represented by mRNA levels in skin biopsies and protein levels in tape strips. Common filaggrin gene mutations were examined in all children. RESULTS: The cytokine profile in lesional skin was dominated by a T helper (Th) 2 response in skin biopsies, and by a general increase in innate inflammation markers (interleukin (IL)-1α, IL-1ß, IL-8, IL-18) along with TARC and CTACK in tape strips. The levels of TARC, CTACK, IL-8, IL-18 showed significant correlation with AD severity in both lesional and non-lesional tape stripped skin, while no significant correlations were observed in skin biopsy data. In tape strips from lesional and non-lesional skin, the levels of NMF and selected cytokines differed significantly between children with and without FLG mutations and food allergy. CONCLUSION: Sampling of the stratum corneum with non-invasive tape strips can be used to identify biomarkers that are associated with disease severity, food allergy and FLG mutations. Skin biopsies showed robust Th2 signature but was inferior for association analysis regarding severity.

Endocrine Disrupting Chemicals and Risk of Testicular Cancer: A Systematic Review and Meta-analysis.

The incidence of many hormone-dependent diseases, including testicular cancer, has sharply increased in all high-income countries during the 20th century. This is not fully explained by established risk factors. Concurrent, increasing exposure to antiandrogenic environmental endocrine disrupting chemicals (EDCs) in fetal life may partially explain this trend. This systematic review assessed available evidence regarding the association between environmental EDC exposure and risk of testicular cancer (seminomas and nonseminomas). Following PRISMA guidelines, a search of English peer-reviewed literature published prior to December 14, 2020 in the databases PubMed and Embase® was performed. Among the 279 identified records, 19 were eligible for quality assessment and 10 for further meta-analysis. The completeness of reporting was high across papers, but over 50% were considered subject to potential risk of bias. Mean age at diagnosis was 31.9 years. None considered effects of EDC multipollutant mixtures. The meta-analyses showed that maternal exposure to combined EDCs was associated with a higher risk of testicular cancer in male offspring [summary risk ratios: 2.16, (95% CI:1.78-2.62), 1.93 (95% CI:1.49-2.48), and 2.78 (95% CI:2.27-3.41) for all, seminoma, and nonseminoma, respectively]. Similarly, high maternal exposures to grouped organochlorines and organohalogens were associated with higher risk of seminoma and nonseminoma in the offspring. Summary estimates related to postnatal adult male EDC exposures were inconsistent. Maternal, but not postnatal adult male, EDC exposures were consistently associated with a higher risk of testicular cancer, particularly risk of nonseminomas. However, the quality of studies was mixed, and considering the fields complexity, more prospective studies of prenatal EDC multipollutant mixture exposures and testicular cancer are needed.

Possible Relevance of Soluble Luteinizing Hormone Receptor during Development and Adulthood in Boys and Men.

Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) are agonists for the luteinizing hormone receptor (LHCGR) which regulates male reproductive function. LHCGR may be released into body fluids. We wish to determine whether soluble LHCGR is a marker for gonadal function. Cross-sectional, longitudinal, and intervention studies on 195 healthy boys and men and 396 men with infertility, anorchia, or Klinefelter Syndrome (KS) were used to correlate LHCGR measured in serum, seminal fluid, urine, and hepatic/renal artery and vein with gonadal function. LHCGR was determined in fluids from in vitro and in vivo models of human testicular tissue and cell lines, xenograft mouse models, and human fetal kidney and adrenal glands. Western blot showed LHCGR fragments in serum and gonadal tissue of similar size using three different antibodies. The LHCGR-ELISA had no species cross-reactivity or unspecific reaction in mouse serum even after human xenografting. Instead, sLHCGR was released into the media after the culture of a human fetal kidney and adrenal glands. Serum sLHCGR decreased markedly during puberty in healthy boys (p = 0.0001). In healthy men, serum sLHCGR was inversely associated with the Inhibin B/FSH ratio (ß -0.004, p = 0.027). In infertile men, seminal fluid sLHCGR was inversely associated with serum FSH (ß 0.006, p = 0.009), sperm concentration (ß -3.5, p = 0.003) and total sperm count (ß -3.2, p = 0.007). The injection of hCG lowered sLHCGR in serum and urine of healthy men (p < 0.01). In conclusion, sLHCGR is released into body-fluids and linked with pubertal development and gonadal function. Circulating sLHCGR in anorchid men suggests that sLHCGR in serum may originate from and possibly exert actions in non-gonadal tissues. (ClinicalTrials: NTC01411527, NCT01304927, NCT03418896).

Using assessment criteria for pesticides to evaluate the endocrine disrupting potential of non-pesticide chemicals: Case butylparaben.

Regulation of chemicals with endocrine disrupting properties depend on the use of the chemical rather than its intrinsic properties. Within the EU, the only criteria currently in place for identifying an endocrine disrupting chemical (EDC) are those developed for biocidal and plant protection products. We argue that ECHA/EFSA guidance for assessing endocrine disrupting properties of biocidal and plant protection products can be applied to all chemicals independent of their intended use. We have assessed the REACH-registered compound butylparaben (CAS 94-36-8), a preservative used primarily in cosmetics. Based on scientific evidence of adverse reproductive effects and endocrine activity, the open literature suggest that butylparaben is an EDC. By applying the ECHA/EFSA guidance for pesticides and biocides, we identify butylparaben as a compound with endocrine disrupting properties. Even though available data is markedly different from that for biocides and pesticides, it was possible to reach this conclusion. More generally, we propose that the ECHA/EFSA guidance can and should be used for identification of EDC regardless of their intended application.

Testicular microlithiasis on scrotal ultrasound in 4850 young men from the general population: associations with semen quality.

BACKGROUND: Testicular microlithiasis (TM) is sometimes found on scrotal ultrasound. The prevalence seems higher in populations of men with testicular dysfunction, and TM may be a risk factor for testicular germ cell neoplasia in situ in men with additional risk factors. The association between TM and testicular function is controversial, especially in incidentally found TM. OBJECTIVES: To determine the frequency of TM in young men from the general population, and associations between TM, semen quality, and reproductive hormones. MATERIALS AND METHODS: A cross-sectional study of 4850 Danish men, median age 19 years. Testicular pattern, including the presence of TM, was assessed by ultrasound examination. Participants provided a questionnaire, one semen sample, and one blood sample. Semen variables and serum reproductive hormones were analyzed as outcomes using multivariable regression analysis to determine associations with TM. RESULTS: TM was detected in 53 men (1%), of which 19 (36%) were unilateral and 34 (64%) were bilateral cases. A history of cryptorchidism was associated with presence of TM. Bilateral TM was associated with slightly lower testicular volume, sperm concentration, and total sperm count. TM was not significantly associated with serum testosterone or other reproductive hormones. DISCUSSION AND CONCLUSION: TM is rare in men from the general population and is associated with lower sperm count if bilateral, although effect sizes were small. Current European guidelines do not recommend any follow-up in cases of TM with no other risk factors for testicular cancer. We suggest that men with incidentally found bilateral TM may be offered a semen analysis, but analysis of reproductive hormones seems unnecessary.

Use of e-cigarettes associated with lower sperm counts in a cross-sectional study of young men from the general population.

STUDY QUESTION: Are use of e-cigarettes and snuff associated with testicular function as previously shown for conventional cigarettes and marijuana? SUMMARY ANSWER: Use of e-cigarettes is associated with reduced semen quality but not with higher serum testosterone level as observed for conventional cigarette use. Snuff use was not associated with markers of testicular function. WHAT IS KNOWN ALREADY: Cigarette smoking has previously been associated with higher testosterone levels and impaired semen quality, whereas it is unresolved whether use of e-cigarettes or snuff influence the testicular function. STUDY DESIGN, SIZE, DURATION: This cross-sectional population-based study included 2008 men with information on cigarette and marijuana use (enrolled between 2012 and 2018), among whom 1221 men also had information on e-cigarette and snuff use (enrolled between 2015 and 2018). PARTICIPANTS/MATERIALS, SETTING, METHODS: Men (median age 19.0 years) from the general population provided a semen and blood sample and filled out a questionnaire on lifestyle including information on smoking behaviour. Associations between different types of smoking (e-cigarettes, snuff, marijuana and cigarettes) and reproductive hormones (total and free testosterone, sex hormone-binding globulin, LH, oestradiol and ratios of inhibin B/FSH, testosterone/LH and free testosterone/LH) and semen parameters (total sperm count and sperm concentration) were examined using multiple linear regression analyses adjusted for relevant confounders. MAIN RESULTS AND THE ROLE OF CHANCE: Approximately half of the men (52%) were cigarette smokers, 13% used e-cigarettes, 25% used snuff and 33% used marijuana. Users of e-cigarettes and marijuana were often also cigarette smokers. Compared to non-users, daily e-cigarette users had significantly lower total sperm count (147 million vs 91 million) as did daily cigarette smokers (139 million vs 103 million), in adjusted analyses. Furthermore, significantly higher total and free testosterone levels were seen in cigarette smoking men (6.2% and 4.1% higher total testosterone and 6.2% and 6.2% higher free testosterone in daily smokers and occasional smokers, respectively, compared to non-smoking men), but not among e-cigarette users. Daily users of marijuana had 8.3% higher total testosterone levels compared to non-users. No associations were observed for snuff in relation to markers of testicular function. LIMITATIONS, REASONS FOR CAUTION: We cannot exclude that our results can be influenced by residual confounding by behavioural factors not adjusted for. The number of daily e-cigarette users was limited and findings should be replicated in other studies. WIDER IMPLICATIONS OF THE FINDINGS: This is the first human study to indicate that not only cigarette smoking but also use of e-cigarettes is associated with lower sperm counts. This could be important knowledge for men trying to achieve a pregnancy, as e-cigarettes are often considered to be less harmful than conventional cigarette smoking. STUDY FUNDING/COMPETING INTEREST(S): Funding was received from the Danish Ministry of Health (1-1010-308/59), the Independent Research Fund Denmark (8020-00218B), ReproUnion (20200407) and the Research Fund of the Capital Region of Denmark (A6176). The authors have nothing to disclose. TRIAL REGISTRATION NUMBER: NA.

Evaluation of Serum Insulin-like Factor 3 Quantification by LC-MS/MS as a Biomarker of Leydig Cell Function.

BACKGROUND: The peptide hormone insulin-like factor 3 (INSL3) is a marker for Leydig cell function and the clinical use of serum INSL3 measurements has been suggested by several groups. AIM: (1) To establish a reference range for liquid chromatography-tandem mass spectrometry (LC-MS/MS) of serum INSL3 in healthy boys and men; and (2) to compare the associations of serum INSL3 and testosterone (T) to pubertal stage, lifestyle factors, diurnal variation, body composition, and human chorionic gonadotropin (hCG) stimulation. RESULTS: In a reference range based on LC-MS/MS analysis of serum from 1073 boys and men, INSL3 increased from levels close to the detection limit (0.03 µg/L) in prepubertal boys to a maximum mean level of 1.3 µg/L (95% CI, 0.9-2.7) in young men (19-40 years of age) and decreased slightly in older men (0.1 µg/L per decade). Serum T, but not INSL3, was associated with body mass index or body fat percentage and with alcohol consumption. Smoking was positively associated with serum T, but negatively associated with INSL3. There were significant diurnal variations in both INSL3 and T in men (P < 0.001), but serum INSL3 varied substantially less, compared with serum T (± 11% vs ± 26%). Mean serum INSL3 increased after hCG stimulation, but less than T (+ 17% vs + 53%). In both healthy men and in patients suspected of testicular failure, baseline serum INSL3 was more closely associated to the hCG-induced increase in serum T than baseline T itself. CONCLUSION: Measurement of serum INSL3 by LC-MS/MS has promise as a marker of testicular disorders.

Associations of Fish Oil Supplement Use With Testicular Function in Young Men.

Importance: Many young men have poor semen quality, and the causes are often unknown. Supplement intake of ω-3 polyunsaturated fatty acid has been found to improve semen quality among men with infertility, but the association with semen quality among healthy men is unknown. Objective: To determine if intake of ω-3 fatty acid supplements is associated with testicular function as measured by semen quality and reproductive hormone levels among healthy men. Design, Setting, and Participants: This cross-sectional study included young Danish men from the general population recruited between January 1, 2012, and December 31, 2017, at compulsory examinations to determine their fitness for military service. Young unselected men were approached after the examination and invited to participate in a study of reproductive function, regardless of their fitness for military service. Data analysis was conducted from September 1, 2018, to June 30, 2019. Exposures: Intake of supplements, including fish oil, during the past 3 months. Main Outcomes and Measures: Semen quality, measured as volume, concentration, total sperm count, percentage of morphologically normal spermatozoa, and motility, and serum reproductive hormone levels, measured as follicle-stimulating hormone, luteinizing hormone, testosterone, free testosterone, and inhibin B levels. Results: Among 1679 young Danish men (median [interquartile range] age, 18.9 [18.7-19.4] years) recruited to participate, 98 men (5.8%) reported use of fish oil supplements during the past 3 months, of whom 53 (54.1%) reported intake on 60 or more days. After adjustment and compared with men with no supplement intake, men with fish oil supplement intake on fewer than 60 days had semen volume that was 0.38 (95% CI, -0.03 to 0.80) mL higher, and men with fish oil supplement intake on 60 or more days had semen volume that was 0.64 (95% CI, 0.15 to 1.12) mL higher (P for trend < .001). Similarly, testicular size in men with supplement intake on fewer than 60 days was 0.8 (95% CI, -0.2 to 1.9) mL larger and in men with fish oil supplement intake on 60 or more days was 1.5 (95% CI, 0.2 to 2.8) mL larger compared with men with no supplement intake (P for trend = .007). After adjustment, men with fish oil supplement intake had a 20% (95% CI, 9%-31%) lower follicle-stimulating hormone level and 16% (95% CI, 8%-24%) lower luteinizing hormone level compared with men with no supplement intake. There were no associations of intake of other supplements with measures of testicular function. Conclusions and Relevance: These findings suggest that intake of fish oil supplements was associated with better testicular function, which is less likely to be due to confounding by indication, as no associations of intake of other supplements with testicular function were found. This cross-sectional study did not examine the actual content of ω-3 fatty acids in the supplements; therefore, these findings need confirmation in well-designed randomized clinical trials among unselected men.

Sex-specific Estrogen Levels and Reference Intervals from Infancy to Late Adulthood Determined by LC-MS/MS.

CONTEXT: The lack of sensitive and robust analytical methods has hindered the reliable quantification of estrogen metabolites in subjects with low concentrations. OBJECTIVE: To establish sex-specific reference ranges for estrone (E1) and estradiol (E2) throughout life and to evaluate sex-differences using the state-of-the-art liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantification of E1, E2, and estriol (E3). DESIGN: LC-MS/MS method development and construction of estrogen reference ranges. SETTINGS: Population-based cross-sectional cohorts from the greater Copenhagen and Aarhus areas. PARTICIPANTS: Healthy participants aged 3 months to 61 years (n = 1838). RESULTS: An isotope diluted LC-MS/MS method was developed and validated for measurements of serum E1, E2, and E3. Limits of detections (LODs) were 3 pmol/L (E1), 4 pmol/L (E2), and 12 pmol/L (E3), respectively. This sensitive method made it possible to differentiate between male and female concentration levels of E1 and E2 in children. In girls, E2 levels ranged from

Inhibition of NFAT Signaling Restores Microvascular Endothelial Function in Diabetic Mice.

Central to the development of diabetic macro- and microvascular disease is endothelial dysfunction, which appears well before any clinical sign but, importantly, is potentially reversible. We previously demonstrated that hyperglycemia activates nuclear factor of activated T cells (NFAT) in conduit and medium-sized resistance arteries and that NFAT blockade abolishes diabetes-driven aggravation of atherosclerosis. In this study, we test whether NFAT plays a role in the development of endothelial dysfunction in diabetes. NFAT-dependent transcriptional activity was elevated in skin microvessels of diabetic Akita (Ins2 +/- ) mice when compared with nondiabetic littermates. Treatment of diabetic mice with the NFAT blocker A-285222 reduced NFATc3 nuclear accumulation and NFAT-luciferase transcriptional activity in skin microvessels, resulting in improved microvascular function, as assessed by laser Doppler imaging and iontophoresis of acetylcholine and localized heating. This improvement was abolished by pretreatment with the nitric oxide (NO) synthase inhibitor l-N G-nitro-l-arginine methyl ester, while iontophoresis of the NO donor sodium nitroprusside eliminated the observed differences. A-285222 treatment enhanced dermis endothelial NO synthase expression and plasma NO levels of diabetic mice. It also prevented induction of inflammatory cytokines interleukin-6 and osteopontin, lowered plasma endothelin-1 and blood pressure, and improved mouse survival without affecting blood glucose. In vivo inhibition of NFAT may represent a novel therapeutic modality to preserve endothelial function in diabetes.

Efferocytosis of Apoptotic Neutrophils Enhances Control of Mycobacterium tuberculosis in HIV-Coinfected Macrophages in a Myeloperoxidase-Dependent Manner.

Tuberculosis remains a big threat, with 1.6 million deaths in 2017, including 0.3 million deaths among patients with HIV. The risk of developing active disease increases considerably during an HIV coinfection. Alveolar macrophages are the first immune cells to encounter the causative agent Mycobacterium tuberculosis, but during the granuloma formation other cells are recruited in order to combat the bacteria. Here, we have investigated the effect of efferocytosis of apoptotic neutrophils by M. tuberculosis and HIV-coinfected macrophages in a human in vitro system. We found that the apo-ptotic neutrophils enhanced the control of M. tuberculosis in single and HIV-coinfected macrophages, and that this was dependent on myeloperoxidase (MPO) and reactive oxygen species in an autophagy-independent manner. We show that MPO remains active in the apoptotic neutrophils and can be harnessed by infected macrophages. In addition, MPO inhibition removed the suppression in M. tuberculosis growth caused by the apoptotic neutrophils. Antimycobacterial components from apoptotic neutrophils could thus increase the microbicidal activity of macrophages during an M. tuberculosis/HIV coinfection. This cooperation between innate immune cells could thereby be a way to compensate for the impaired adaptive immunity against M. tuberculosis seen during a concurrent HIV infection.

Possible link between FSH and RANKL release from adipocytes in men with impaired gonadal function including Klinefelter syndrome.

INTRODUCTION: The FSH receptor (FSHR) has been found to be expressed in human bone cells and bone marrow-adipocytes, and highly-debated mouse studies have suggested extra-gonadal effects of gonadotropins on glucose, adipocyte and bone homeostasis. These putative effects could be direct or indirectly mediated by endocrine factors released from bone-cells or adipocytes. Here, we investigated whether gonadotropins are linked with glucose- and lipid-metabolism in hypergonadotropic men. METHODS: Single centre, cross-sectional study of 307 men with idiopathic infertility and 28 men with Klinefelter syndrome (KS). OUTCOME: associations between serum LH and FSH with soluble-RANKL (sRANKL), osteoprotegerin (OPG), osteocalcin, fasting glucose and insulin, sex steroids, and body composition. Expression of FSHR was studied in human-derived adipocyte-cell-models (hMADS, TERT-hWA) and FSH stimulation of RANKL expression and secretion in hMADS in vitro. RESULTS: Serum FSH was not directly linked with glucose- and lipid-metabolism. However, FSH was inversely associated with sRANKL in both infertile men and KS men (p = .023 and p = .012). Infertile men with elevated FSH (>11 U/L) had significantly lower sRANKL (p = .015). sRANKL was positively associated with fat percentage, fasting insulin, and glucose (all p < .05). Men with prediabetes had higher sRANKL (p = .021), but lower testosterone (p < .0001) and Inhibin B (p = .005). The FSHR was expressed in the investigated human derived adipocytes, and 3-6 h treatment with FSH markedly increased RANKL release (p < .05). CONCLUSION: KS and infertile men with prediabetes have low Inhibin B, and testosterone but elevated RANKL compared with non-prediabetic men despite comparable levels of serum gonadotropins. Serum FSH and sRANKL was inversely associated in both infertile and KS men, but the increased release of RANKL from FSH treated adipocytes suggest a direct effect of FSH on RANKL production in some tissues. Further studies are required to clarify whether FSH targets RANKL in the skeleton. ClinicalTrial_ID:NCT01304927.

Cross-sectional analysis of sleep hours and quality with sex hormones in men.

BACKGROUND: Reduced total hours of sleep and low quality of sleep have been suggested to be associated with low levels of male hormones. Few studies have examined the association between excessive sleep and male reproductive hormones. OBJECTIVE: To investigate the association of total hours of sleep and quality of sleep with serum levels of total, bioavailable and free testosterone (tT, bT and fT), sex hormone-binding globulin (SHBG) and dehydroepiandrosteron-sulfate (DHEAS). METHODS: Serum levels of tT, SHBG and DHEAS were measured with immunoassays in a cross-sectional population-based study of 2095 males. bT and fT were calculated in accordance with Vermeulens method. Information on total hours of sleep and sleep quality was obtained by questionnaire. Linear regression was used to calculate hormones according to total hours of sleep and the results were expressed as ß-estimates and 95% confidence intervals (CI). The adjustment in the multivariable models was constructed taking age, BMI, smoking, alcohol intake and physical activity into account. RESULTS: Excessive sleep (>9 h) compared to 7-9 h of sleep was significantly associated with lower tT, bT and fT, but not with SHBG or DHEAS, after multivariable adjustment. These significant associations were also found in our analyses with hormones as continuous variables but no associations were found in our general additive model analyses. CONCLUSIONS: In this cross-sectional study in men, excessive sleep associated with lower levels of male reproductive hormones. Longitudinal studies are needed to determine the causal direction of the observed association between excessive sleep and lower male reproductive hormones levels.