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The plasticizer di(2-ethylhexyl) phthalate (DEHP) is known to have endocrine-disrupting properties mediated by its many metabolites that form upon exposure in biological systems. In a previous study, we reported an inverse association between DEHP metabolites in the human ovarian follicular fluid (FF) and the responsiveness of the follicles to controlled ovarian stimulation during in vitro fertilization (IVF) treatments. Here, we explored this association further through molecular analysis of the ovarian FF samples.Ninety-six IVF patients from Swedish (N = 48) and Estonian (N = 48) infertility clinics were selected from the previous cohort (N = 333) based on the molar sum of DEHP metabolites in their FF samples to arrive at “high” (mean 7.7 ± SD 2.3 nM, N = 48) and “low” (0.8 ± 0.4 nM, N = 48) exposure groups. Extracellular miRNA levels and concentrations of 15 steroid hormones were measured across FF samples. In addition, FF somatic cells, available for the Estonian patients, were used for RNA sequencing.Differential expression (DE) and interactions between miRNA and mRNA networks revealed that the expression levels of genes in the cholesterol biosynthesis and steroidogenesis pathways were significantly decreased in the high compared to the low DEHP group. In addition, the DE miRNAs were predicted to target key enzymes within these pathways (FDR

Background: Studies suggest a link between pregnancy phthalate exposures and gestational diabetes mellitus (GDM). Few studies have evaluated associations between phthalate biomarkers (individual or mixtures) with gradations of maternal glucose intolerance. Methods: In a subset of 606 women participating in LIFECODES pregnancy cohort, a combination of 50-gram 1-h non-fasting glucose load test (GLT) and 100-gram 3-h fasting oral glucose tolerance test was used to determine pregnancy glycemic status (median: 27 weeks gestation): normoglycemia (n = 136), impaired glucose tolerance (IGT) (n = 296), and GDM (n = 174). Nineteen metabolites of phthalates and their replacements were measured during each trimester. We used multivariable logistic regression models to evaluate associations between biomarkers (in quartiles) and maternal glycemic status (GDM v. normoglycemia and IGT v. normoglycemia), adjusting for potential confounders. We also used principal component analysis to evaluate associations jointly accounting for metabolites as chemical mixtures. Results: Higher 1st trimester mono-3-carboxypropyl phthalate (MCPP) was associated with decreased odds of GDM (Q4 v. Q1: 0.30; 95% CI: 0.13, 0.67) and IGT (Q4 v. Q1 OR: 0.37; 95% CI: 0.17, 0.79). Higher 2nd trimester mono-isobutyl phthalate (MiBP) was associated with increased IGT (Q4 v. Q1 OR: 2.07; 95% CI: 1.06, 4.07), and 2nd trimester mono-3-hydroxybutyl phthalate (MHBP) was non-monotonically associated with increased GDM (Q2 v. Q1 OR: 3.21; 95% CI: 1.54, 6.87). Mixture analyses showed similar associations (Q4 v. Q1 for 2nd trimester dibutyl phthalates metabotlites mixtures OR: 2.08; 95% CI: 1.04, 4.22). Conclusion: Some phthalate biomarkershad trimester-specific associations with glycemic outcomes, with long and short term health implications.

Background: We have previously investigated whether urinary concentrations of bisphenol A (BPA), parabens, and phthalate metabolites were individually associated with reproductive outcomes among women undergoing in vitro fertilization (IVF) treatment. However, humans are typically exposed to many man-made chemicals simultaneously. Thus, investigating one chemical at a time may not represent the effect of mixtures. Objective: To investigate whether urinary concentrations of BPA, parabens, and phthalate metabolite mixtures are associated with reproductive outcomes among women undergoing IVF. Methods: This prospective cohort study included 420 women contributing 648 IVF cycles who provided up to two urine samples per cycle prior to oocyte retrieval (N = 1145) between 2006 and 2017 at the Massachusetts General Hospital Fertility Center, and had available urine biomarker data. Urinary concentrations of BPA, parabens, and phthalate metabolites were quantified using isotope-dilution tandem mass spectrometry. Intermediate and clinical end-points of IVF treatments were abstracted from electronic medical records. Principal component analysis (PCA) and Bayesian kernel machine regression (BKMR) were used to identify main patterns of BPA, parabens, and phthalate metabolites concentrations. We used generalized linear mixed models to evaluate the association between PCA-derived factor scores, in quartiles, and IVF outcomes, using random intercepts to account for multiple IVF cycles and adjusting for known confounders. Because of temporal trends in exposure, we conducted a sensitivity analysis restricted to women who underwent IVF cycles in the earlier years of study (2006–2012). Results: Urinary concentrations of BPA, parabens, and most phthalate metabolites were significantly lower during the second half of the study period (2013–2017) than during the first half (2006–2012). None of the three factors derived from the PCA [di(2-ethylhexyl) phthalate (DEHP), non-DEHP, and paraben] was associated with IVF outcomes in the main analyses. Similarly, BKRM analyses did not identify any associations of individual urinary concentrations of BPA, paraben and phthalate metabolites with IVF outcomes while accounting for correlation between exposures. However, in sensitivity analyses restricted to women who underwent IVF cycles from 2006 to 2012, where concentrations of most phthalates and phenols were higher, there were decreases in implantation, clinical pregnancy, and live birth across quartiles of the DEHP factor. Specifically, women in the highest quartile of the DEHP factor had, on average, lower probabilities of implantation (−22% p, trend = 0.08), clinical pregnancy (−24% p, trend = 0.14), and live birth (−38% p, trend = 0.06) compared to women in the lowest quartile. Among this group of women, BKMR results did not identify any single contributor driving the decreased probabilities of live birth within the DEHP factor. Conclusions: We confirmed that women undergoing IVF are concurrently exposed to multiple endocrine disrupting chemicals (EDCs). While we found no overall significant associations, we observed diminished pregnancy success with specific clusters of chemicals among women who underwent IVF cycles in earlier years of study, when urinary concentrations of these EDCs were higher.

Background: Higher blood lead levels (BLLs) have been linked to neurologic deficits and impaired growth, but few studies have evaluated their association with timing of sexual maturity or pubertal progression in boys. Methods: In a longitudinal cohort of Russian boys enrolled at age 8–9 and followed to adulthood, BLLs were measured at study entry, and pubertal staging (genitalia and pubic hair) and testicular volume (TV) measurements were obtained annually. We used interval-censored regression models to estimate differences between boys with higher BLL (≥5 μg/dL) and lower BLL in mean ages at sexual maturity (genitalia stage 5, pubic hair stage 5, or TV ≥ 20 mL) and duration of pubertal progression (onset to maturity), adjusting for potential confounders. Mediation analyses were conducted to quantify the percent of lead's effect attributable to its association with reduced somatic growth. Results: Among 481 evaluable boys, 28% had BLL ≥ 5 μg/dL. Adjusted mean ages at sexual maturity were 14.7 years for genitalia, 16.1 for pubic hair, and 13.9 for TV. In adjusted models, boys with BLLs ≥5 μg/dL had later maturity than those with lower levels by 4–5 months depending on pubertal indicator. In mediation analyses, height and body mass index at age 11 accounted for 40–71% of the shift in age at maturity for boys with higher compared to lower BLLs. Higher BLLs were not associated with pace of pubertal progression. Conclusions: Higher lead levels were associated with later attainment of sexual maturity in males, but not with the duration of pubertal progression. A high proportion of the delay in sexual maturity for boys with higher as compared to lower BLL was shown to be attributable to mediating effects of BLL on reduced growth. Keywords: Puberty, Growth, Sexual maturity, Blood lead, Endocrine disruptors, Interval-censoring, Mediation analysis, Pubertal progression

Background: Few epidemiologic studies have evaluated the impact of paternal environmental exposures, particularly as mixtures, on couples’ pregnancy outcomes. Objective: We investigated whether mixtures of paternal urinary bisphenol A (BPA), paraben, and phthalates were associated with pregnancy outcomes among couples attending a fertility center. Methods: We included 210 couples undergoing 300 in vitro fertilization (IVF) between 2004 and 2017 in this prospective analysis. We quantified paternal urinary biomarker concentrations in one sample per cycle using isotope-dilution tandem mass spectrometry. We used principal component analysis (PCA) to identify correlations of biomarker concentrations and multivariable Cox proportional hazards models for discrete survival time to estimate the hazard ratios (HRs) and 95% CIs for the associations between PCA-derived factor scores and probability of failing to achieve a live birth. Interactions were also included in the models to examine strength of associations over three vulnerable periods [embryo transfer to implantation, implantation to clinical pregnancy, and clinical pregnancy to live birth]. Models were adjusted for paternal and maternal ages and body mass indexes, urinary dilution (specific gravity) and year of collection, infertility diagnosis, and other PCA factor scores. Sensitivity analyses with further adjustment for maternal PCA factor scores were performed. Results: We identified three factors, representing di-2-ethylhexyl phthalate (DEHP) metabolites, BPA and non-DEHP metabolites, and parabens, accounting for 56%, 15% and 10%, respectively, of the total variance explained. An interquartile range (25th and 75th percentiles) increase in the DEHP-related factor score was associated with elevated probability of failing prior to live birth (HR = 1.41, 95% CI: 1.08, 1.81) and the association was stronger between implantation and clinical pregnancy as well as between clinical pregnancy and live birth compared to before implantation. The overall HRs of failure for the BPA/non-DEHP-related and paraben-related factor scores were HR = 1.24 (95% CI: 0.97, 1.59) and HR = 0.99 (95% CI: 0.80, 1.24). We found similar HRs when additionally adjusting for maternal PCA factor scores. Conclusion: Paternal mixtures of urinary concentrations of DEHP metabolites were related to higher infertility treatment failure.

Mediation analysis offers an essential and rapidly expanding tool in environmental health studies to investigate the contribution of environmental factors towards observed associations between risk factors and health outcomes. When evaluating environmental factors, there may be particular interest in quantifying the impact of exposure to environmental mixtures on human health. In this context, evaluating the joint effect of multiple chemicals or pollutants, rather than individual examination, allows accurate identification of risk factors, assessment of interactions, and ultimately development of more targeted public health interventions. While mediation analysis has been extended to incorporate several methodological complexities specific to environmental factors, little attention has been given to integrating the analysis of environmental mixtures.The aim of this review is to present some of the available methods for environmental mixtures, and discuss how these methods can be integrated within a mediation analysis framework. By incorporating these methods into a mediation framework, investigators will be able to evaluate the contribution of environmental mixtures as mediators of exposure-outcome associations, based on methodologies that are currently available.While standard regression-based methods for multiple mediators can be used, these can easily become unstable as the number of mixture components increases. Summary and classification methods, or hierarchical modeling, can reduce the number of mediators by creating scores or possibly uncorrelated subgroups. This approach allows retrieving indirect effects due to the mixture or to a specific subgroup, but makes identification of component-specific effects and interactions complicated. Finally, one can use various approaches for analyzing mixtures in a two-stage fashion, selecting relevant mediators to be included in the final model.We focused this review on techniques that have been presented to the environmental health community and that can be conducted with major statistical software. We encourage researchers to move beyond the evaluation of one environmental factor at a time to the assessment of the joint effects of environmental mixtures when a mediation model is of interest. Available methods target different aspects related to environmental mixtures and the choice of the suitable approach will depend on data structures and the research question of interest. Keywords: Environmental mixtures, Environmental epidemiology, Methods, Mediation analysis