Sex-specific associations between maternal exposure to parabens, phenols and phthalates during pregnancy and birth size outcomes in offspring.

Current evidence on the effects of prenatal exposure to endocrine disrupting chemicals on birth size remains largely inconclusive. We aimed to investigate sex-specific associations between maternal exposure to parabens, phenols and phthalates during pregnancy and birth weight, length and head/abdominal circumferences. We performed a prospective study of 88 pregnant women who underwent amniocentesis in the period 2012 to 2014. Maternal urine samples were collected during pregnancy in weeks 12 to 36 (median: 18 weeks). The concentrations of parabens, phenols and individual phthalate diester metabolites were analyzed by isotope-diluted liquid chromatography-tandem mass spectrometry and osmolality adjusted. Linear regression models estimated the associations between urinary levels of selected compounds (tertile(T2-T3)medium/high versus T1low exposure) and birth size, stratified by offspring sex. A total of three parabens, two phenols, four individual phthalate metabolites and four sums of diester metabolites were detectable above limits of detection in at least 60% of urine samples. Overall, we observed few statistically significant associations, but medium/high exposure to bisphenol A (BPA) in male offspring was associated with statistically significant lower birth size across most outcomes [birth weight: -428 g (95% CI -756 to -99.4); birth length: -1.76 cm (95% CI -3.28 to -0.25); abdominal circumference: -1.97 cm (95% CI -3.55 to -0.39)]. Similarly, medium/high exposure to methyl paraben (MeP) in male offspring was associated with lower birth weight (-661 g, 95% CI -1251 to -70.7) and length (-3.11 cm, 95% CI -5.76 to -0.46) compared to low exposure. None of these associations were statistically significant in female offspring. Across all compounds, individual exposures were associated with more negative estimates of birth weight for male than for female offspring. Our study indicates that prenatal exposure to BPA and MeP may negatively affect birth size outcomes, with a possible sex effect. Given the small sample size, these findings need to be replicated in future larger studies.

Presence of parabens, phenols and phthalates in paired maternal serum, urine and amniotic fluid.

OBJECTIVE: To examine whether selected endocrine disrupting chemicals were present in pregnant women and passed through the placental barrier to amniotic fluid, potentially exposing the developing fetus. METHODS: Paired samples of maternal serum, urine and amniotic fluid were concurrently collected (<1 h) from 200 pregnant women (age >18 years) with a singleton pregnancy and undergoing amniocentesis between gestational weeks 12 - 36. The concentration of six different parabens, seven phenols, 31 metabolites from 15 phthalate diesters and the polychlorinated substance triclocarban were analyzed by isotope diluted TurboFlow-liquid chromatography-tandem mass spectrometry. RESULTS: Concentrations of all included compounds were highest in maternal urine followed by serum, and lowest in amniotic fluid. Of the six parabens measured in amniotic fluid, methylparaben (MeP) and ethylparaben (EtP) were detectable most often (87% and 33% of the samples, respectively). Of the seven phenols measured, three (2,4-dichlorphenol, 2,5-dichlorphenol, 2-propylphenol) were detectable in the range of 14-21% of the amniotic fluid samples, at low concentrations (<0.12 ng/ml). Two secondary phthalates metabolites, mono-(2-carboxymethyl-hexyl) phthalate and mono-carboxy-iso-octyl phthalate were each present in ≤15% of the amniotic fluid samples at concentrations 2-5 times lower than in maternal serum and 20-100 times lower than in maternal urine. A modest statistically significant correlation between the levels of MeP and EtP was detected in paired maternal urine-amniotic fluid samples was detected (Spearman rMeP: 0.246; rEtP: 0.364). Likewise, the concentration of mono-ethyl phthalate (MEP) in paired maternal urine and amniotic fluid samples indicated a modest statistically significant correlation (Spearman rMEP: 0.264), driven by detectable levels of MEP in only 3% of the amniotic fluid samples. CONCLUSIONS: In general, the included parabens, phenols and phthalates were effectively metabolized and excreted via the urine, which was the matrix that reflected the highest detectable levels. The detectable levels of several included parabens and phthalates in human amniotic fluid calls for further investigations of the toxicokinetic and potential endocrine disrupting properties of individual and multiple endocrine disruptors in order to better assess the risk to the developing fetus.

UV filters in matched seminal fluid-, urine-, and serum samples from young men.

Recent in vitro studies have shown that some chemical UV filters mimic the effect of progesterone in the activation of the CatSper Ca2+ channel in human spermatozoa. However, so far, the extent of exposure of human spermatozoa to chemical UV filters via the presence of these chemicals in seminal fluid has been unknown. Here, we present levels of UV filters measured in human seminal fluid and comparisons to levels measured in concurrently collected urine and serum samples. In total nine UV filters were analysed by TurboFlow-LC-MS/MS in paired urine, serum, and seminal fluid samples from 300 young Danish men from the general population; each man collected one of each sample type within 1 h. The samples were collected during February-December 2013 and only six of the men reported having used sunscreen during the 48 h preceding the sample collection. Four of the examined UV filters could be detected in seminal fluid samples at levels above LOD in more than 10% of the samples. Benzophenone (BP), benzophenone-1 (BP-1), and benzophenone-3 (BP-3) were most frequently detected in, respectively, 18%, 19%, and 27% of the seminal fluid samples albeit at levels one to two orders of magnitude lower than the levels observed in urine. 4-methyl-benzophenone (4-MBP) was detectable in 11% of the seminal fluid samples while in <5% of the urine samples. Overall 45% of the men had at least one of the UV filters present in their seminal fluid at detectable levels. For BP-1 and BP-3 individual levels in urine and seminal fluid were significantly correlated, while this was not evident for BP nor 4-MBP. In conclusion, chemical UV filters are present in men's seminal fluid; some of which can activate the human sperm-specific CatSper Ca2+ channel and thereby potentially interfere with the fertilisation process.

FSHB and FSHR gene variants exert mild modulatory effect on reproductive hormone levels and testis size but not on semen quality: A study of 2020 men from the general Danish population.

BACKGROUND: Spermatogenesis depends on stimulation by follicle-stimulating hormone (FSH) which binds to FSH receptors (FSHR) on testicular Sertoli cells. Three FSH-related single-nucleotide polymorphisms (SNPs), FSHB -211G>T (rs10835638), FSHR -29G>A (rs1394205) and FSHR 2039A>G (rs6166) affect FSH action, and have been suggested to affect testicular function, but the evidence is uncertain. OBJECTIVE: To describe the associations between the three SNPs and testicular function in a large and well-characterised cohort of men from the general population. MATERIALS AND METHODS: A cross-sectional study of 2020 Danish men unselected regarding testicular function. Outcome variables were semen parameters, reproductive hormones and testis size. Genotyping was done by competitive allele-specific quantitative PCR. Differences in genotype frequencies were tested by chi-square test and associations between genotypes and outcomes were assessed by multivariate linear regressions. RESULTS: The SNPs affected serum FSH; carriers of the variant affecting FSH secretion (FSHB -211G>T) had lower FSH levels while carriers of variants affecting receptor expression (FSHR -29G>A) and receptor sensitivity (FSHR 2039A>G) had higher FSH levels. Carriers of FSHB -211G>T had lower calculated free testosterone/LH ratio. Although both FSHB -211G>T and FSHR 2039A>G were associated with smaller testis size, no clear association was detected in relation to any semen parameters, except a lower total number of morphologically normal spermatozoa in the heterozygous carriers of the FSHB -211G>T DISCUSSION AND CONCLUSION: The studied polymorphisms have only minor modulating influence on testis size and function in healthy men. We detected subtle effects of the three SNPs on FSH levels, but also effects of FSHB -211G>T on calculated free testosterone/LH ratio, compatible with altered Leydig cell function. Thus, the role of these FSH-related polymorphisms is complex and modest in men with normal testicular function, but the possible importance of FSH polymorphisms in men with impaired testicular function should be evaluated in future studies in more detail.

Use of stored serum in the study of time trends and geographical differences in exposure of pregnant women to phthalates.

BACKGROUND: Exposure to some phthalate diesters has been associated with adverse reproductive health outcomes in both rodents and humans indicative of anti-androgenic effects. Exposure during sensitive periods of development, such as prenatally, is of particular concern. OBJECTIVES: We wished to investigate whether phthalate metabolites measured in maternal serum samples from historical birth cohorts can be used to assess prenatal exposure. Further, we aimed to study temporal and geographical trends in phthalate exposure across three different birth cohorts. METHODS: We compared phthalate metabolite levels in maternal serum samples from an Australian (1989-91) and a Danish (1997-2001) birth cohort with levels in serum and urine samples from a recent Danish birth cohort (2012-14). Samples were analysed for 32 phthalate metabolites from 15 phthalate diesters by isotope-diluted liquid chromatography-tandem mass spectrometry (LC-MS/MS). Correlations between metabolites were tested by Spearman rank correlation test, and differences between the cohorts were tested by Mann-Whitney U test. RESULTS: Overall, we observed large variations in serum phthalate metabolite levels between individuals. Secondary metabolites of di-(2-ethyl-hexyl) phthalate (DEHP) and di-iso-nonyl phthalate (DiNP) in serum were weakly to moderately and positively correlated to the levels measured in urine, and secondary metabolites of DEHP were also moderately to strongly and significantly correlated in serum. Correlations with mono-(2-ethyl-hexyl) phthalate (MEHP) and mono-iso-nonyl phthalate (MiNP), the two primary metabolites of DEHP and DiNP, were inconsistent, and we found indications of sample contamination. We observed some significant differences in phthalate metabolite levels between the three cohorts with generally higher levels in the older birth cohorts. CONCLUSION: Based on comparison across two older birth cohorts and a recent cohort, our results support the concept that historical biobanked serum samples may be used for assessment of prenatal exposure to phthalates when using serum levels of the monoesters of the low-molecular weight (LMW) phthalates and the secondary metabolites of the high-molecular weight (HMW) phthalates. Serum phthalate measurements are, however, not suitable for human biomonitoring and should only be used to exploit historical samples from cohorts, where urine samples were not collected. Our findings suggest that phthalate exposure may have decreased over time from the early 1990s to the 2010s.

Exposure to UV filters during summer and winter in Danish kindergarten children.

BACKGROUND: Ultra violet (UV) filters with known or suspected endocrine disrupting properties are widely used in sunscreens and other personal care products, clothing, food packaging and many other consumer products. Danish kindergarten children have sunscreens applied daily during summer to prevent skin burns. OBJECTIVES: To estimate the assumed contribution of sunscreens to the total exposure to UV filters, we measured the urinary excretion of UV filters during summer and winter in kindergarten children. METHODS: Spot- and first morning urines were collected during a summer and a winter day in 2013. A total of 266 urine samples were collected from 55 children and were analysed for content of benzophenone (BP), benzophenone-1 (BP-1), benzophenone-2 (BP-2), benzophenone-3 (BP-3), 5-chloro-2-hydroxybenzophenone (BP-7), 4-methyl-benzophenone (4-MBP), 4-hydroxybenzophenone (4-HBP), 3-(4-methylbenzylidene)-camphor (4-MBC), and 3-benzylidene camphor (3-BC) by LC-MS/MS. RESULTS: Of the analysed UV filters, the children excreted predominantly BP-1, BP-3 and 4-HBP. The urine levels were significantly higher in summer samples compared to winter samples, however exposure during winter was still evident. Furthermore, children with the highest concentrations of UV filters in summer urines also tended to be among those with the highest winter levels. CONCLUSION: Exposures to UV filters during summertime can partly be explained by the intended use of UV filters in sunscreens, which is considered to be beneficial for children during outdoor activities. However, exposure to UV filters all year round together with large inter-individual variation indicate that children's exposure to UV filters also comes from other consumer items, presumably highly influenced by the general lifestyle of an individual child: this is completely unintended, without benefit, and potentially harmful.

Self-reported onset of puberty and subsequent semen quality and reproductive hormones in healthy young men.

STUDY QUESTION: Is there an association between pubertal onset and subsequent reproductive health in young men? SUMMARY ANSWER: Self-reported later onset of puberty was associated with reduced semen quality and altered serum levels of reproductive hormones among 1068 healthy, young Danish men. WHAT IS KNOWN ALREADY: The long-term effects of variations in the onset of male puberty on subsequent reproduction remain largely unstudied. STUDY DESIGN, SIZE, DURATION: In a cross-sectional study, young healthy Danish men were approached when they attended a compulsory medical examination to determine their fitness for military service from 2008 to 2012. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: A total of 1068 healthy, young Danish men (mean age 19 years) participated. They were asked to assess whether onset of penile and testicular growth, development of pubic hair and voice break occurred earlier, at the same time as or later than their peers. Their semen quality (semen volume, sperm concentration, total sperm count and percentages of motile and morphologically normal spermatozoa) and serum concentrations of sex hormones (LH, FSH, total testosterone, SHBG, inhibin B) and testicular size were determined. MAIN RESULTS AND THE ROLE OF CHANCE: The response rate was 29%. Of the 1068 men who then participated, 652 answered the questions about penile growth and pubic hair development and were therefore included in the analysis. Self-reported later onset of puberty was associated with a 25% reduction in sperm concentration (95% CI -41%; -4%), a 40% reduction in total sperm count (-55%; -21%), a 1.6% age point reduction in morphological normal spermatozoa (-2.9; -0.3) and a 1.6 ml reduction in testicular size (-2.4 and -0.8 ml), after adjustment for confounders. Self-reported later onset of puberty was also associated with a 9% (3%; 15%) reduction in free testosterone and a 16% (2%; 31%) increase in FSH, after adjustment for confounders. LIMITATIONS, REASON FOR CAUTION: Our study was cross-sectional and reverse causality cannot be ruled out. In addition, we cannot rule out the possibility that the men with late puberty onset had not yet fully matured although most were in Tanner stage 5. WIDER IMPLICATIONS OF THE FINDINGS: Approximately 15% of young Danish men have self-reported later onset of puberty than their peers. We found poorer testicular function in young men with a history of later pubertal development, suggesting that timing of pubertal onset may be a fundamental marker of male reproductive health. However, we cannot exclude the possibility that these men had not fully matured at the time of examination and therefore their semen quality may yet improve, which makes follow-up important. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the Danish Council for Strategic Research, Program Commission on Health, Food and Welfare (project number 2101-08-0058), Rigshospitalet (grants 961506336 and R42-A1326), European Union, DEER (grant agreement no 212844), the Danish Ministry of Health and the Danish Environmental Protection Agency and Kirsten and Freddy Johansens Foundation (grant 95-103-72087). There are no competing interests.