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In Utero and Peripubertal Metals Exposure in Relation to Reproductive Hormones and Sexual Maturation and Progression among Boys in Mexico City
Deborah Watkins
University of Michigan School of Public Health
Corresponding Author
ORCiD: https://orcid.org/0000-0002-6692-2206
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Background
Endocrine disrupting chemicals (EDCs) such as metals have been reported to alter circulating reproductive hormone concentrations and pubertal development in animals. However, the relationship has rarely been investigated among humans, with the exception of heavy metals, such as Pb and Cd. Our aim was to investigate measures of in utero and peripubertal metal exposure in relation to reproductive hormone levels and sexual maturation and progression among boys from the Early Life Exposure in Mexico to Environmental Toxicants (ELEMENT) cohorts.
Methods
Our analysis included 118 pregnant women and their male children from the ELEMENT study. Essential and non-essential metals were measured in urine collected from the mothers during the third trimester of pregnancy and their male children at 8-14 years. Reproductive hormone levels serum testosterone, estradiol, dehydroepiandrosterone sulfate (DHEA-S), inhibin B, and sex hormone-binding globulin (SHBG) were measured in blood samples from the children at 8-14 years. We also assessed Tanner stages for sexual maturation (genital, pubic hair development, and testicular volume), at two time points (8–14, 10-18 years). We used linear regression to independently examine urinary metal levels in relation to each peripubertal reproductive hormones adjusting for child age and BMI. Generalized estimation equations (GEEs) were used to evaluate the association of in utero and peripubertal metal exposures with sexual maturation and progression during follow-up based on Tanner staging and testicular volume.
Results
In utero and prepubertal concentrations of some urinary metals were associated with increased levels of peripubertal reproductive hormones, especially non-essential metal(loid)s As and Cd ( in utero ), and Ba (peripubertal) as well as essential metal Mo ( in utero ) in association with testosterone. More advanced pubic hair developmental stage and higher testicular volume at the early teen visit was observed for boys with higher non-essential metal concentrations, including in utero Al and peripubertal Ba, and essential metal Zn concentration (peripubertal). These metals were also associated with slower pubertal progression between the two visits.
Conclusion
These findings suggest that male reproductive development may be associated with both essential and non-essential metal exposure during in utero and peripubertal windows.
Keywords
Metal, Hormone, In utero exposure, Pregnancy, Puberty
Figure 1
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Figure 3
In recent decades, a trend toward earlier onset of puberty among boys and girls has been described (Anderson et al. 2003; Euling et al. 2008; Eveleth 2017; Herman-Giddens et al. 1997; Herman-Giddens et al. 2001; Karpati et al. 2002; Lee et al. 2016; Paris et al. 2016; Wacharasindhu 2017; Wyshak and Frisch 1982). These trends in the timing of puberty, a period of physical and psychological development, have raised concerns regarding the potential impact of environmental factors, including endocrine disrupting chemicals (EDCs) (Blakemore et al. 2010; Buck Louis et al. 2008; Lee and Styne 2013; Sisk and Foster 2004). Exposure to EDCs in early life and at the prepubertal stage are thought to play a role in altered pubertal timing, possibly via their estrogenic or anti-androgenic effects and disruption of normal homeostatic control of the hypothalamic-pituitary-gonadal (HPG) or hypothalamic-pituitary-adrenal (HPA) axis (Bellingham et al. 2009; Buck Louis et al. 2008; Den Hond and Schoeters 2006; Diamanti-Kandarakis et al. 2009; Fudvoye et al. 2019; Jacobson-Dickman and Lee 2009; Jansen et al. 2018; Y. Liu et al. 2019; Massart et al. 2006; Parent et al. 2016; Pescovitz and Walvoord 2007; Rodriguez et al. 2019; Roy et al. 2009; Watkins et al. 2016; Watkins et al. 2017).
Some metals and metalloids, such as cadmium (Cd), lead (Pb), mercury (Hg), and arsenic (As), are non-essential xenobiotics that are known to be harmful to human health (Atsdr 2007a, b; Faroon et al. 2012; Ha et al. 2017). These non-essential metal(loid)s are persistent in the environment and children’s exposure to them is nearly ubiquitous (Claus Henn et al. 2014; World Health Organization 2017). Several other metals, such as chromium (Cr), copper (Cu), manganese (Mn), molybdenum (Mo), selenium (Se) and zinc (Zn), are essential for optimal health but may be toxic at insufficient or excessive levels (Gerberding 2005; Greger 1999; Health and Services 2004; Risher 2003; Shenkin 2003). A number of metal(loid)s are reproductive toxicants and have endocrine disrupting properties which interfere with many aspects of hormone action (Bloom et al. 2010; De Coster and van Larebeke 2012; Deb and Mandal 2017; Diamanti-Kandarakis et al. 2009; Mendiola et al. 2011; Wallace 2019; Yilmaz et al. 2019).
Many human and animal studies have focused on elucidating the reproductive effects associated with heavy metal and metalloids, such as Cd, Pb, Hg, and As. Though many studies on Cd were either cross-sectional and/or conducted on adults, there has been some consistency of findings with regard to the positive relationship between male and female Cd exposure and testosterone levels (Bochud et al. 2017; Garcia-Morales et al. 1994; Jurasovic et al. 2004; Meeker et al. 2010; Menke et al. 2008; Nagata et al. 2005; Telisman et al. 2007; Zeng et al. 2002; Zeng et al. 2004). In a group of men with no occupational exposure, positive associations between blood Pb concentrations and testosterone and/or estradiol levels were reported (Jurasovic et al. 2004; Meeker et al. 2010; Telisman et al. 2007). In longitudinal studies of children’s reproductive development, childhood Pb exposure was related to later pubertal onset in Russian boys was in Chapaevsk, Russia (Hauser et al. 2008; Williams et al. 2010; Williams et al. 2019). In contrast, we have shown early life exposure to Pb was associated with delays in pubertal development in girls but not boys in Mexico City (Jansen 2018, Liu 2019). Hg was found to be associated with increased estradiol levels in both males and females from a small residential population in Cambodia (Agusa et al. 2007), which was in agreement with a previous study among women with repeated miscarriages (Gerhard et al. 1998). A recent study of As exposure through well water consumption in Taiwan suggested that arsenic may impart an increased risk of erectile dysfunction through a reduction of circulating testosterone levels (Hsieh et al. 2008).
Although less attention has been given to the other metals in the past, a growing body of evidence suggests that certain essential or trace metals, including Cu, fluoride (F), Mn, Mo, and Se can also have adverse effects on male reproduction (Jeng et al. 2015; Yun Liu et al. 2019; Meeker et al. 2008; Telisman et al. 2000). In addition, most reports on the male reproductive effects of metals are from experimental animal, epidemiological, and occupational studies usually involving high doses not commonly encountered by children. Due to the widespread exposure of humans and known adverse effects related to essential and non-essential metal exposure, concern is growing that low-level exposure may also adversely affect reproductive developmental outcomes in boys. Moreover, only a few studies have investigated the cross-sectional relationships in boys, with the exception of two recent longitudinal studies in Russia and Mexico (Yun Liu et al. 2019; Williams et al. 2019), and none have examined exposure during in utero development and subsequent hormone levels during puberty, a time at which steroid hormones play an essential role in reproductive development (Cahill 2006; Parent et al. 2015; Sisk and Foster 2004; Spear 2000).
Therefore, the present study assessed whether in utero and prepubertal exposure to metals at relatively low doses altered reproductive hormone levels or timing and progression of sexual maturation in boys. We extended the limited metals studied on this topic and examined both essential and non-essential metals in relation to reproductive hormone levels and progression of sexual maturation in boys from ages 8–14 years to 10–18 years.
2.1. Study population
Participants in this study are part of the “Early Life Exposure in Mexico to Environmental Toxicants (ELEMENT)” project, a longitudinal cohort study of pregnant women in Mexico City and their children (Perng et al. 2019). Our analysis included women who were recruited from maternity hospitals during their first trimester between 1997 and 2004 and their children, as previously described (Ashrap et al. 2019; Lewis et al. 2013; Y. Liu et al. 2019; Perng et al. 2019; Wu et al. 2018). Mothers completed interview-based questionnaires at up to three prenatal visits [mean gestational age at visit 1: 13.5 (range:9–24) weeks, visit 2: 25.1 (range: 19–37) weeks, visit 3: 34.4 (range: 28–43) weeks] and provided spot urine samples at the third trimester visit. Between 2008 and 2011, a subset of their children (n = 250, 132 girls and 118 boys), who were then 8–14 years of age, were selected based on the availability of archived maternal biological specimens and re-contacted to participate in follow-up studies (i.e. early-teen visit). From those 250 children, 223 (89%) were enrolled again when they were age 10–18 years (late adolescence/adult) in a second follow-up study (i.e. late-teen visit), in 2013–2017 (Ashrap et al. 2019). Among those 223 children, 109 were boys (92% retention rate from early to late-teen visit) and the average follow up period was 3.5 years. Participants provided spot urine and fasting blood samples, anthropometry, and reported socio-demographic information via an interviewer-administered questionnaire. In the current analyses, we included male children who finished the early-teen visit (a majority of these same boys also completed late-teen visit) and had maternal urinary metal concentration measurements and/or their early-teen visit urinary metal measurements available (n = 118).
2.2. Metal Concentrations
Urinary metal concentrations of 14 metals and metalloids: aluminum (Al), As, barium (Ba), Cd, cobalt (Co), Cu, iron (Fe), Mn, Mo, nickel (Ni), Pb, antimony (Sb), selenium (Se), and Zn were measured in maternal third trimester urine and urine samples collected during the early-teen visit at age 8–14 years. Prenatal and peripubertal (early-teen) urine samples were collected in sterile cups, aliquoted within one hour after collection, frozen and stored at -80°F, and shipped on dry ice to McGill University (Montreal, Canada) for analysis. Urinary metals were measured using inductively coupled plasma mass spectrometry (ICPMS; Varian 820-MS, Inc., Palo Alto, California) as described previously (Basu et al. 2010; Srigboh et al. 2016). Accuracy and precision were measured using certified reference standards (Institut National de Santé Publique du Québec, or INSPQ) with coefficients of variation (CVs) ranging from 3 to 14%, and each batch run contained procedural blanks and replicate runs (Lewis et al. 2018; Srigboh et al. 2016). More details regarding quality control (QC) were previously described (Lewis et al. 2018). Values below the limit of detection (LOD) were replaced with the LOD/√2. Urinary specific gravity (SG) was measured using a handheld digital refractometer.
Pb exposure in this study was also measured in maternal patella/blood and early peripubertal blood, and results of these biomarkers in relation to pubertal development within this population have already been published (Jansen et al. 2018; Y. Liu et al. 2019). As patella and blood Pb levels are better biomarkers of long term Pb exposure, we excluded urinary Pb from the current analyses.
2.3. Hormones
Children provided fasting blood samples during the early-teen visit at age 8–14 years. Serum aliquots were separated and frozen at − 80 °C, and then sent to the Clinical Ligand Assay Service Satellite (CLASS) Laboratory at the University of Michigan (Ann Arbor, MI) for hormone analysis. Estradiol, testosterone, inhibin B, and sex hormone-binding globulin (SHBG) were measured in serum samples as biomarkers of puberty, and dehydroepiandrosterone sulfate (DHEA-S) was measured as a biomarker of adrenarche. Estradiol, total testosterone, SHBG, and DHEA-S were measured using an automated chemiluminescent immunoassay (Bayer Diagnostics ACS:180). Active inhibin B was assayed using Gen II ELISA (Beckman Coulter, Webster, TX). Values below the LOD were replaced with the LOD/√2.
2.4 Sexual Maturation
Two pediatricians evaluated male offspring for Tanner staging and testicular volume using standardized protocols during both follow-up visits at ages 8–14 and 10–18 years. To ensure consistency, pediatricians were trained prior to the start of each follow-up as previously described (Chavarro et al. 2017) to evaluate Tanner staging in male participants using standardized protocols. Genital stage (GD) was assessed as an indicator of puberty and pubic hair stage (PH) as an indicator of adrenarche, with stage 1 corresponding to no development and stage 5 corresponding to full development (Marshall and Tanner 1969). Right and left testicular volume (TV) were measured with an orchidometer, and the larger of the two measurements was used in analyses. As a testicular volume of 1–3 mL is considered prepubertal (Ankarberg-Lindgren and Norjavaara 2004; Mouritsen et al. 2013) and TV ≥ 20 mL was used as an indicator of sexual maturity (Ankarberg-Lindgren and Norjavaara 2004; Sergeyev et al. 2017), cutoffs of 3 mL and 20 mL were used to create a 3-level ordinal variable for testicular volume.
2.5 Covariates
Covariates in our analysis included: age at the early-teen visit, BMI z-scores at both visits, and household socioeconomic status at the late-teen visit. BMI z-scores were calculated based on the World Health Organization child reference curves for age and sex (WHO, 2007) for each follow up visit. A 7-level categorical variable for socioeconomic status (SES) was estimated using a validated scale consisting of thirteen questions on housing quality, services, material goods and head of household education (Asociación Mexicana de Agencias de Investigación de Mercados y Opinión Pública, AMAI version 13 × 6) (AMAI 2000; López 2008).
2.5. Statistical Methods
The geometric mean concentrations of each metal in urine samples from pregnant women and their children were calculated. The percent of urine samples with concentrations below the LOD were reported for each metal, and metals that were detected in less than 50% of samples were excluded from further analysis.
Multiple linear regressions were used to assess associations between urinary metal concentrations and peripubertal serum hormone concentrations, where serum hormones were natural log-transformed prior to analysis to achieve normal distribution. Metal concentrations from prenatal and peripubertal urine samples were entered into regression models separately and each model was adjusted for child age and BMI z-score, and for SG as a measure of urinary dilution. Results were calculated as the percent difference in hormone (95% confidence interval) per interquartile range (IQR) increase in urinary metal concentrations. Because reproductive hormones may differ greatly in boys at pre-pubertal stages (Tanner stage = 1) vs pubertal stages (Tanner stage > 1), we performed a secondary analysis in which we examined the associations between in utero and peripubertal exposures and reproductive hormone levels among subjects who were pre-pubertal (pubic hair Tanner stage = 1, n = 94).
Longitudinal analyses were conducted to explore the association between metals and early-teen and late-teen sexual maturation parameters using repeated measures generalized estimating equation (GEE). This allows us to take fuller advantage of the data that have been collected our longitudinal cohort study and use the additional within-person information to achieve increases in statistical power to detect associations (Sitlani et al. 2015). With separate models for each metal, the GEE approach was used to fit a multinomial (ordinal) regression model for pubertal stages at each visit as a function of metal exposure, age at early-teen visit and change in time, with adjustment for potential confounders:
g(E[Yij]) = β0 + β1Mij + β2Agei + β3Timeij + β4Mij*Timeij + β5Agei*Timeij (1)
g(E[Yij]) = β0 + β1Mij + β2Agei + β3Timeij + β4Mij*Timeij + β5Agei*Timeij
+β 6 BMIbase + β6 BMIvar (2)
Model (1) is the crude model where Y is an outcome of interest (Tanner stages/testicular volume), g is a link function (cumulative logit), i denotes subject number (1,…,n), and j denotes visit number (1, 2). M is ln-transformed metal exposure, A is the age at the early-teen visit, Time is the change in time between the early-teen and late-teen visit. Age at early-teen visit and change in time between two visits were included in the model to account for the effect of baseline age on attained Tanner stage or testicular volume, the natural pubertal progression across time, and the effect of baseline age on the natural pubertal progression. Model (2) is the final adjusted model, in which BMI z-score at the early-teen visit (BMIbase) and the change in BMI z-score from early-teen to late-teen visit (BMIvar) were included. The coefficients of interest are the cross-sectional effect of metal on tanner stage/testicular volume (β1) and the effect of metal on the progression of tanner stage/testicular volume over time (β4). Given the possibility that age and BMI z-score at the early-teen visit may be associated with Tanner stage and influence the future progression of Tanner stage, working independence was chosen as the covariance structure to ensure the validity of parameter estimates (Sullivan Pepe and Anderson 1994). We ran the GEE models both with and without BMI z-score (model 1 and 2) as BMI may be on the causal pathway between exposure and puberty. However, the magnitude of estimates from both models were almost identical, therefore we reported results from the models including BMI z-score. Results are presented as odds ratios (OR) and 95% confidence intervals (95% CI) per IQR increase in exposure. As few participants were categorized as having a PH Tanner stage = 3 and stage = 4 during the early-teen visit, the number of covariates we could reliably enter into models was limited (Peduzzi et al. 1996; Vittinghoff and McCulloch 2007). Thus, to minimize the number of covariates in GEE models, we included SG-corrected metal concentrations (rather than entering SG as a separate covariate which yielded similar results), using the following equation: Pc = P[(SGp – 1)/(SGi – 1)] where Pc is the SG corrected metal concentration (µg/L), P is the measured metal concentration, SGp is the median urinary specific gravity, and SGi is the individual’s urinary specific gravity.
Because SES could be a potential confounder, we ran a sensitivity analysis including SES as a covariate in models of hormones and maturation stages. Furthermore, as both low and high levels of essential metals are of concern, to explore potential non-linear associations we used adjusted generalized additive models (GAM) to graphically depict the cross-sectional relationship between metal concentrations and hormones and sexual maturation measurements. We also considered significance after adjusting for multiple testing using the Benjamini-Hochberg method (Benjamini and Hochberg 1995). Since Tanner stages/testicular volumes were not independent of each other, we calculated q values (adjusted p values) treating each outcome as a family of tests (11 tests per outcome). A cutoff of 0.15 for q value was used to further interpret main results with greater confidence. All analyses were performed using R version 3.5.2 and SAS 9.4.
3.1. Exposure and Outcome Distributions
The percent of samples with metal concentrations below the limit of detection, as well as the geometric means, standard deviations, and selected percentiles of sample concentrations from prenatal and peripubertal (early-teen) visits are shown in Table 1. Spearman correlations between the prenatal and peripubertal visit metal concentrations adjusted for SG are also presented in the table. With the exception of Sb and Fe, other metals were detected in > 50% of urine samples, therefore, Sb and Fe were excluded from further analysis. Weak correlations between maternal and peripubertal metal levels and weak to moderate correlations between different urinary metal concentrations within maternal and child samples have been previously reported elsewhere (Lewis et al. 2018). Distributions of Tanner stages of sexual maturation and testicular volume among the male children at the two follow up visits are reported (SI Table S1). At early -teen visit, the majority of the boys (n = 94, 81.7%) were at Tanner stage 1 for pubic hair development whereas 57 boys (49.6%) were at Tanner stage 1 for genital development. At the late-teen visit, boys moved to more advanced Tanner stages for sexual measurement- only 28 (26.4%) and 9 (7.5%) were at Tanner stage 1 for pubic hair development and genital development and 14 (13.2%) and 18 (13.2%) reached full development (Tanner stage = 5) for the two measurements. In terms of testicular volume distribution, the percentage of the boys in the prepubertal stage dropped from 14.8–0% from early-teen to late-teen visit.
| In utero | Peripubertal | |||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| LOD | %< LOD | GM | GSD | 25% | 50% | 75% | MAX | %< LOD | GM | GSD | 25% | 50% | 75% | MAX | P value b | |
| Essential metals | ||||||||||||||||
| Co | 0.4 | 0.0 | 1.2 | 1.9 | 0.8 | 1.2 | 2.1 | 5.2 | 0.0 | 0.7 | 1.5 | 0.6 | 0.8 | 0.9 | 1.6 | 0.63 |
| Cu | 48.2 | 46.3 | 86.1 | 4.4 | 34.1 | 50.2 | 71.9 | 2742 | 56.8 | 43.3 | 1.3 | 34.1 | 34.1 | 56.1 | 106 | 0.83 |
| Mn | 0.4 | 7.4 | 0.8 | 1.7 | 0.6 | 0.7 | 1.0 | 8.7 | 2.5 | 1.2 | 1.8 | 0.8 | 1.2 | 1.7 | 4.4 | 0.68 |
| Mo | 2.9 | 15.8 | 19.5 | 3.3 | 12.7 | 25.7 | 42.9 | 308 | 0.0 | 46.6 | 1.8 | 33.7 | 50.2 | 67.1 | 210 | 0.99 |
| Se | 10.5 | 3.2 | 29.9 | 1.7 | 23.0 | 32.6 | 43.1 | 120 | 1.7 | 47.2 | 1.7 | 36.3 | 53.0 | 65.6 | 141 | 0.26 |
| Zn | 0.1 | 1.2 | 271 | 2.2 | 160 | 298 | 451 | 1253 | 0.0 | 366 | 1.8 | 257 | 411 | 521 | 1200 | 0.08 |
| Non-essential Metals | ||||||||||||||||
| Al | 8.6 | 11.6 | 24.0 | 2.5 | 12.3 | 20.3 | 42.2 | 304 | 24.6 | 14.5 | 2.1 | 8.8 | 14.0 | 23.3 | 428 | 0.37 |
| As | 0.3 | 0.0 | 14.0 | 2.0 | 9.3 | 13.2 | 20.6 | 153 | 0.0 | 14.3 | 2.0 | 10.2 | 14.4 | 20.5 | 515 | 0.07 |
| Ba | 1.1 | 3.2 | 4.0 | 2.0 | 2.6 | 4.2 | 5.9 | 27.6 | 10.2 | 2.5 | 2.0 | 1.6 | 2.4 | 3.9 | 20.2 | 0.81 |
| Ni | 3.0 | 0.0 | 8.7 | 1.9 | 5.8 | 7.4 | 11.0 | 107 | 0.8 | 8.1 | 1.6 | 5.9 | 8.0 | 10.8 | 53.2 | 0.83 |
| Cd | 54.0 | 2.1 | 0.2 | 2.1 | 0.1 | 0.2 | 0.3 | 2.7 | 2.5 | 0.1 | 1.6 | 0.1 | 0.1 | 0.2 | 0.3 | 0.24 |
| a uncorrected for specific gravity | ||||||||||||||||
| b P value from Spearman correlation test between in utero and peripubertal metal concentration measurements. | ||||||||||||||||
| a Results are presented as the percent difference in peripubertal hormone levels associated with an interquartile range (IQR) increase in in utero and peripubertal metal | ||||||||||||||||
| b Linear regression models were adjusted for child age, BMI z-score and specific gravity | ||||||||||||||||
| * Significant associations with p value < 0.05 & q value < 0.15 | ||||||||||||||||
| * Significant associations with p value < 0.05 & q value > 0.15 | ||||||||||||||||
| a GEE models were adjusted for child age and BMI z-score (baseline and change) | ||||||||||||||||
| * Significant associations with p value < 0.05 & q value < 0.15 | ||||||||||||||||
| * Significant associations with p value < 0.05 & q value > 0.15 | ||||||||||||||||
| a GEE models are adjusted for child age and BMI z-score (baseline and change) | ||||||||||||||||
| * Significant associations with p value < 0.05 & q value < 0.15 | ||||||||||||||||
| * Significant associations with p value < 0.05 & q value > 0.15 | ||||||||||||||||
| Supplemental Information | ||||||||||||||||
| In Utero and Peripubertal Metal(loid)s Exposure in Relation to Reproductive Hormones and Sexual Maturation and Progression among boys in Mexico City | ||||||||||||||||
| Pahriya Ashrap, John D. Meeker, Brisa N. Sánchez, Niladri Basu, Marcela Tamayo-Ortiz, Maritsa Solano-González, Adriana Mercado-García, Martha M. Téllez-Rojo, Karen E. Peterson, Deborah J. Watkins* | ||||||||||||||||
| Table of Contents: | ||||||||||||||||
3.2 In utero and peripubertal metal exposure and peripubertal hormone levels
Associations between in utero and peripubertal metal concentrations and reproductive hormones are presented in Fig. 1 and SI Table S3. Positive associations were observed between some urinary essential metal concentrations and estradiol, testosterone, and SHBG. One IQR increase in in utero Zn concentration was associated with 13.7% higher serum estradiol levels (95% CI: 0.3, 28.8). In utero Co and Mn were positively associated with SHBG levels, with an IQR increase associated with 16% (95% CI:0.4, 34.2) and 14.2% (95%CI: 1.5, 28.5) higher serum SHBG after adjustment for child age, BMI z-score, and SG, respectively. As shown in Fig. 1, effect estimates for the association between both in utero essential and non-essential metal concentrations and testosterone were larger compared to other reproductive hormones, 51.3% for Mo, 35% for As, 38.9% for Cd. In models where reproductive hormones were regressed on concurrent peripubertal exposures, the strongest associations were observed also between metal concentrations and testosterone, particularly with non-essential metals Ni (%△/IQR: 35.1%, 95% CI: 2.6, 77.8) and Ba (%△/IQR: 59.1%, 95% CI: 22.5, 106.8). Peripubertal Ba concentration was also associated with higher estradiol (%△/IQR: 10.2%, 95% CI: 0.5, 20.9). However, no significant associations were detected between peripubertal metal exposures and DHEA-S or inhibin B.
After correcting for multiple testing, the associations of in utero Mo, As, and Cd with testosterone levels, as well as the association of peripubertal Ba with testosterone level had q-values < 0.15 (Fig. 1 and SI Table S3), providing greater confidence in these associations.
3.3 In utero and peripubertal metal exposure and sexual maturation
We have presented results from multiple ordinal regression models of in utero metal concentrations and Tanner stage and testicular volume in Fig. 2 and SI Table S4. Tanner stages and testicular volume at the early-teen visit were not associated with in utero metal concentrations, with the exception of a non-essential metal, Al; an IQR increase in in utero Al concentrations was associated with 3.6 times greater odds (95% CI: 1.67, 7.76) of being at a higher category of testicular volume versus lower categories. In the same figure and table, associations between in utero metal concentrations and pubertal development over time in boys are also presented. During the follow-up, an IQR increase in in utero concentrations of non-essential metalloid As was associated with 36% (OR/IQR: 0.64, 95% CI: 0.48, 0.85) lower odds of genital development progression per year, adjusting for age, BMI, and Tanner stage at the early-teen visit (GEE longitudinal model). In utero concentrations of non-essential metal(loid)s Al (OR/IQR: 0.61, 95% CI: 0.45, 0.83) and As (OR/IQR: 0.64, 95% CI: 0.43, 0.97) were associated with lower odds of progressing to a higher testicular volume category (i.e. 39 and 36% lower odds/IQR). Essential metal Zn was also associated with 38% lower odds of testicular volume progression (OR/IQR: 0.62, 95% CI: 0.44, 0.88).
Similarly, Fig. 3 and SI Table S4 show the cross-sectional and longitudinal associations between prepubertal metal concentrations and sexual maturation and progression. No significant associations were found between peripubertal urinary metal concentrations with Tanner stage for genital development or progression over follow-up, although several essential and non-essential metals were associated with pubic hair Tanner stages, testicular volume, and progressions. Peripubertal Zn concentration was associated with higher odds of being at a higher developmental stage for pubic hair (OR/IQR: 6.11, 95% CI: 1.89, 19.69) and testicular volume (OR/IQR: 5.39, 95% CI: 1.88, 15.49) at the early-teen visit, as well as slower progression of pubic hair development (OR/IQR: 0.47, 95% CI: 0.34, 0.67) and testicular volume (OR/IQR: 0.58, 95% CI: 0.40, 0.85) during the follow-up. Higher Mn was associated with 34% (OR/IQR: 0.66, 95% CI: 0.46, 0.96) lower pubic hair development progression only. Regarding non-essential metals, an IQR increase in peripubertal Ba concentration was associated with 2.3 times greater odds (95% CI: 1.15, 4.75) of being at higher Tanner stage for pubic hair development at age 8–14 years, but with 34% (OR/IQR: 0.66, 95% CI: 0.53, 0.83) lower odds of progressing to a higher pubic hair Tanner stage per year of follow-up. Higher peripubertal urinary Al was also associated with 47% (OR/IQR: 0.53, 95% CI: 0.40, 0.70) lower odds of pubic hair development progression per IQR increase.
All the associations described above had q-values < 0.15 (Fig. 2, Fig. 3, and SI Table S4) after correcting for multiple testing, except for the association between in utero As and testicular volume progressions. Output graphics produced from GAM models showed that there was no significant non-linear relationship between essential and non-essential metal concentrations and hormones and sexual maturation outcomes, after adjusting for the same sets of covariates.
3.4 Sensitivity Analysis
Results from the hormone subset analysis when we restricted to boys who were prepubertal (94 out of 118) at the early-teen visit are similar to the main analysis results (SI Table S5). For models regressing in utero metal concentrations, some effect estimates for DHEA_S, SHBG, and testosterone were no longer significant, potentially due to the small sample size. For models regressing peripubertal exposure, the notable effect estimates for testosterone were similar to those from the main analysis. The association between DHEA_S and Ni (%△/IQR = 20.2, 95%CI = 1.7, 42.0) and inhibin B and Al (%△/IQR =-19.2, 95%CI=-31.7, -4.4) became stronger and significant in the peripubertal subset analysis.
The magnitude of estimates from GEE models with and without BMI (SI Table S6) were almost identical. Findings from models adjusting for SES were generally consistent with metal and hormone associations observed in our main analyses; in SES-adjusted models, the association between in utero Co concentrations and higher SHBG (%△/IQR = 14.2, 95%CI=-2.9, 23.3) was slightly attenuated and no longer significant, while the association between peripubertal Al and inhibin B (%△/IQR=-14.1, 95%CI=-24.6, -2.2) was stronger and significant. In GEE models for Tanner stage or testicular volume status including SES finding remain consistent with the main models.
A high proportion of samples had metal concentrations above the LOD except for Cu, which was below the detection limit in 46% of samples. In a secondary analysis, we categorized urinary Cu concentrations into three groups. The low group consisted of values below the LOD, while the medium and high groups were made up of equalized bins among the detected values. We estimated the model parameters again and found that they were similar to the main parameter estimates.
Previous studies of metal exposure and male reproductive development have primarily been cross-sectional and focused on heavy metals, with the exception of “the Russian Children's Study” and “Early Life Exposure in Mexico to Environmental Toxicants (ELEMENT) Study” that assessed the longitudinal impact of blood Pb (Yun Liu et al. 2019; Williams et al. 2019). Thus, it is critical to evaluate the associations of essential and non-essential metals with reproductive hormones, sexual maturation, and progression among boys. Based on our longitudinal analysis of boys in a birth cohort in Mexico City who were examined at two peripubertal follow-up visits, we demonstrated a higher pubic hair development stage and testicular volume at the early-teen visit with higher non-essential metal concentrations, including in utero Al and peripubertal Ba and essential metal Zn concentration (peripubertal). We also found associations between in utero and peripubertal exposure to a number of both essential and non-essential metals and slowed progression of pubertal development across the follow-up period. In relation to hormone levels, higher non-essential metals, in utero As and Cd and peripubertal Ba concentrations as well as essential metal Mo measured in utero were associated with substantially higher testosterone levels. None of the associations between metals and hormones and measurements of sexual maturations appear to be non-linear.
To our knowledge, this is the first study to investigate the association between various in utero and peripubertal metals measured in urine and reproductive hormone levels in teenage boys. We reported a number of positive associations between specific metals measured in the urine and reproductive hormones, particularly testosterone. Some of our findings have not been reported elsewhere, such as relationships of essential minerals, e.g., Zn with estradiol or Co and Mn with SHBG. The positive association between relatively low Cd exposure and testosterone in this study is supported by epidemiological studies of non-occupational exposures among men (Jurasovic et al. 2004; Meeker et al. 2010; Menke et al. 2008; Telisman et al. 2007). However, in vivo and in vitro observations linked Cd with altered adrenal gland activity and induced oxidative stress, which could result in toxic effects on the testis and subsequently decrease in serum testosterone (Gay et al. 2013; Liu et al. 2009; Patra et al. 2011; Singhal et al. 1976; Siu et al. 2009).
Mo is an essential nutrient that is a cofactor for important enzymes involving in toxin removal (Schwarz 2016; Todd et al. 2017). While no prior studies have investigated the associations between Mo and hormones in boys, contrary to our finding on in utero Mo and increased testosterone, decreased testosterone levels and sperm concentrations were observed in males with increasing levels of Mo (Lewis and Meeker 2015; Meeker et al. 2008; Meeker et al. 2010). Several animal studies have also reported the reproductive toxicity of Mo, including declines in sperm concentration, motility and normal morphology, testicular degeneration, and reduced fertility (Jeter and Davis 1954; Pandey and Singh 2002; Thomas and Moss 1951; Vyskočil and Viau 1999). It is worth noting that the prenatal Mo levels were lower in our study population compared to the previous studies of Mo and reproductive outcomes and those reported in the National Health and Nutrition Examination Survey (NHANES) (Centers for Disease Control and Prevention 2018) (SI Table S2). It is not clear whether Mo levels measured in utero will have different effects on reproductive hormones measured during different life stages, which warrants further investigation. Although previous studies have not evaluated associations between Ba and reproductive hormones in human, our finding of positive associations between peripubertal Ba concentrations and serum testosterone and estradiol levels is consistent with previous studies among male zebrafish, where exposure to barium chloride significantly increased estradiol concentrations and transcripts of genes involved in the HPG axis (Kwon et al. 2016).
One of the main observations in this study is that Zn is associated with higher odds of pubic hair and testicular volume stage at the early-teen follow-up, but a slower progression of puberty from the early-teen to late-teen visit. Few cross-sectional studies on boys and girls also reported a significant positive relationship between serum or plasma zinc levels and stages of sexual maturation (Leonard et al. 1998; Onukwuli et al. 2018; Vivoli et al. 1990; Wagner et al. 1985), as well as reproductive hormones (testosterone). Zn is an essential trace metal and is fundamental for the development of the male reproductive system (Kerns et al. 2018; Mendoza et al. 2017). Studies on various animals described the role of Zn in advancing male puberty, through increased testicular activity, testosterone production, metabolism, and growth, primarily via activated the hypothalamus and the pituitary functions (Arangasamy et al. 2018; Dance et al. 2016; El-Masry et al. 1994; Fang and Furuhashi 1978; Geary et al. 2016; Underwood and Somers 1969). However, we did not observe a significant association between serum Zn and testosterone levels.
Pubic hair stages at the early-teen visit and progression were consistently associated with elevated concentrations of certain peripubertal metals, including As, Al, Mn, and Zn. The biological mechanisms that underlie the link between these essential and non-essential metals and pubertal development are uncertain but may be related to reproductive hormones. The main hormones responsible for pubic hair development in males are the androgens DHEA and testosterone (Breehl and Caban 2020; Rubinow and Schmidt 1996). In this cohort, the strongest observed associations were between in utero and peripubertal metal concentrations and increased testosterone, while no significant associations were seen in relation to DHEA-S. It is possible that these metals impact the appearance of pubic hair through changes in testosterone.
In this study, we observed slower progression of sexual maturation for those boys at higher Tanner stages at the early-teen visit which was also reported in studies explored the association between pubertal onset and progression (German et al. 2018; Llop-Viñolas et al. 2004; Pantsiotou et al. 2008). A compensatory mechanism similar to “catch-up growth” was proposed previously as a potential explanation for this observation (Ashrap et al. 2019; Wu et al. 2018). For those boys who had experienced a delayed pubertal development, their body systems may have responded by accelerating the tempo of pubertal progression (the change from lower stages of puberty to higher stages); while others who had experienced an advanced pubertal development may respond by slowing down the pubertal progression. This concept may explain the associations between metal exposure, earlier puberty onset at early-teen, and slower progression we found in this study. Future research is needed to establish this phenomenon and potential mechanisms.
The ELEMENT study, a prospective longitudinal birth cohort study in Mexico City, provided an opportunity to study the relationships of both in utero and peripubertal metal exposure on hormone levels and measures of sexual maturation and progression during the peripubertal period. Our results indicate that the in utero and peripubertal periods are vulnerable life stages, during which metal exposures may lead to disruption of male reproductive hormones and pubertal development. The findings also support that essential and non-essential metals have the potential to disrupt the onset and progression of puberty via interrupting the critical hormonal pathways. Several of the associations with hormones and sexual maturation are comparable to previous reports from human and animal research and supported by the current understanding of male pubertal development. However, there are inconsistent findings between the current study and previous studies and this may due to a number of reasons; 1) the exposure assessment approaches were different [sampling period, exposure matrices (i.e., blood, urine, hair)]. 2) most of the previous studies exploring the relationship between metals and hormones were conducted on adult population. 3) most studies examined the cross-sectional relationships whereas we examined the longitudinal association between metals and sexual maturation and progression. Limitations of our study include a somewhat small sample size and few observations for certain Tanner stages, which may result in imprecise effect estimates. Some of the metals measured in this study have a relatively short half-life in urine, so urinary levels at the time measurement may not fully characterize exposure during each specific window of development. Lastly, hormone levels were only measured at one time point and are likely to be subject to non-differential misclassification due to diurnal variation. Further research is warranted to prospectively explore the underlying mechanisms by which metals may affect male sexual maturation and progression in larger study populations.
Ethics approval and consent to participate
Research protocols were approved by the Institutional Review Board at University of Michigan, and the Research, Biosafety and Ethics in Research at the Mexico National Institute of Public Health. Prior to enrollment, informed consent from mothers and informed assent from offspring were obtained.
Consent for publication
Not applicable.
Availability of data and materials
All data generated and analyzed during this study are not publicly available due to the Institutional Review Board restrictions.
Competing interests
The authors declare that they have no competing interests.
Funding
This work was supported by U.S. Environmental Protection Agency (US EPA) grants RD834800 and RD83543601 and National Institute for Environmental Health Sciences (NIEHS) grants P20 ES018171, P01 ES02284401, and P30 ES017885. Its contents are solely the responsibility of the grantee and do not necessarily represent the official views of the US EPA. Further, the US EPA does not endorse the purchase of any commercial products or services mentioned in the publication. This work was also supported and partially funded by the National Institute of Public Health, Ministry of Health of Mexico.
Authors’ Contributions
PA conducted data analyses and drafted the manuscript. JDM, KEP, MTR and DJW conceived the study and oversaw its coordination. NB helped oversee the study, data acquisition, metals analysis, and advised the interpretation and drafting of the manuscript. BNS helped oversee the study and advised during the data analysis. MSG and AMG participated in the collection and assembly of data. All authors read and approved the final manuscript.
Acknowledgments
The authors acknowledge the research staff at participating hospitals and the American British Cowdray Hospital in Mexico City for providing research facilities. We thank the mothers and children for participating in the study.
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In Utero and Peripubertal Metals Exposure in Relation to Reproductive Hormones and Sexual Maturation and Progression among Boys in Mexico City
Deborah Watkins
University of Michigan School of Public Health
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ORCiD: https://orcid.org/0000-0002-6692-2206
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Background
Endocrine disrupting chemicals (EDCs) such as metals have been reported to alter circulating reproductive hormone concentrations and pubertal development in animals. However, the relationship has rarely been investigated among humans, with the exception of heavy metals, such as Pb and Cd. Our aim was to investigate measures of in utero and peripubertal metal exposure in relation to reproductive hormone levels and sexual maturation and progression among boys from the Early Life Exposure in Mexico to Environmental Toxicants (ELEMENT) cohorts.
Methods
Our analysis included 118 pregnant women and their male children from the ELEMENT study. Essential and non-essential metals were measured in urine collected from the mothers during the third trimester of pregnancy and their male children at 8-14 years. Reproductive hormone levels serum testosterone, estradiol, dehydroepiandrosterone sulfate (DHEA-S), inhibin B, and sex hormone-binding globulin (SHBG) were measured in blood samples from the children at 8-14 years. We also assessed Tanner stages for sexual maturation (genital, pubic hair development, and testicular volume), at two time points (8–14, 10-18 years). We used linear regression to independently examine urinary metal levels in relation to each peripubertal reproductive hormones adjusting for child age and BMI. Generalized estimation equations (GEEs) were used to evaluate the association of in utero and peripubertal metal exposures with sexual maturation and progression during follow-up based on Tanner staging and testicular volume.
Results
In utero and prepubertal concentrations of some urinary metals were associated with increased levels of peripubertal reproductive hormones, especially non-essential metal(loid)s As and Cd ( in utero ), and Ba (peripubertal) as well as essential metal Mo ( in utero ) in association with testosterone. More advanced pubic hair developmental stage and higher testicular volume at the early teen visit was observed for boys with higher non-essential metal concentrations, including in utero Al and peripubertal Ba, and essential metal Zn concentration (peripubertal). These metals were also associated with slower pubertal progression between the two visits.
Conclusion
These findings suggest that male reproductive development may be associated with both essential and non-essential metal exposure during in utero and peripubertal windows.
Figure 1
Figure 2
Figure 3
In recent decades, a trend toward earlier onset of puberty among boys and girls has been described (Anderson et al. 2003; Euling et al. 2008; Eveleth 2017; Herman-Giddens et al. 1997; Herman-Giddens et al. 2001; Karpati et al. 2002; Lee et al. 2016; Paris et al. 2016; Wacharasindhu 2017; Wyshak and Frisch 1982). These trends in the timing of puberty, a period of physical and psychological development, have raised concerns regarding the potential impact of environmental factors, including endocrine disrupting chemicals (EDCs) (Blakemore et al. 2010; Buck Louis et al. 2008; Lee and Styne 2013; Sisk and Foster 2004). Exposure to EDCs in early life and at the prepubertal stage are thought to play a role in altered pubertal timing, possibly via their estrogenic or anti-androgenic effects and disruption of normal homeostatic control of the hypothalamic-pituitary-gonadal (HPG) or hypothalamic-pituitary-adrenal (HPA) axis (Bellingham et al. 2009; Buck Louis et al. 2008; Den Hond and Schoeters 2006; Diamanti-Kandarakis et al. 2009; Fudvoye et al. 2019; Jacobson-Dickman and Lee 2009; Jansen et al. 2018; Y. Liu et al. 2019; Massart et al. 2006; Parent et al. 2016; Pescovitz and Walvoord 2007; Rodriguez et al. 2019; Roy et al. 2009; Watkins et al. 2016; Watkins et al. 2017).
Some metals and metalloids, such as cadmium (Cd), lead (Pb), mercury (Hg), and arsenic (As), are non-essential xenobiotics that are known to be harmful to human health (Atsdr 2007a, b; Faroon et al. 2012; Ha et al. 2017). These non-essential metal(loid)s are persistent in the environment and children’s exposure to them is nearly ubiquitous (Claus Henn et al. 2014; World Health Organization 2017). Several other metals, such as chromium (Cr), copper (Cu), manganese (Mn), molybdenum (Mo), selenium (Se) and zinc (Zn), are essential for optimal health but may be toxic at insufficient or excessive levels (Gerberding 2005; Greger 1999; Health and Services 2004; Risher 2003; Shenkin 2003). A number of metal(loid)s are reproductive toxicants and have endocrine disrupting properties which interfere with many aspects of hormone action (Bloom et al. 2010; De Coster and van Larebeke 2012; Deb and Mandal 2017; Diamanti-Kandarakis et al. 2009; Mendiola et al. 2011; Wallace 2019; Yilmaz et al. 2019).
Many human and animal studies have focused on elucidating the reproductive effects associated with heavy metal and metalloids, such as Cd, Pb, Hg, and As. Though many studies on Cd were either cross-sectional and/or conducted on adults, there has been some consistency of findings with regard to the positive relationship between male and female Cd exposure and testosterone levels (Bochud et al. 2017; Garcia-Morales et al. 1994; Jurasovic et al. 2004; Meeker et al. 2010; Menke et al. 2008; Nagata et al. 2005; Telisman et al. 2007; Zeng et al. 2002; Zeng et al. 2004). In a group of men with no occupational exposure, positive associations between blood Pb concentrations and testosterone and/or estradiol levels were reported (Jurasovic et al. 2004; Meeker et al. 2010; Telisman et al. 2007). In longitudinal studies of children’s reproductive development, childhood Pb exposure was related to later pubertal onset in Russian boys was in Chapaevsk, Russia (Hauser et al. 2008; Williams et al. 2010; Williams et al. 2019). In contrast, we have shown early life exposure to Pb was associated with delays in pubertal development in girls but not boys in Mexico City (Jansen 2018, Liu 2019). Hg was found to be associated with increased estradiol levels in both males and females from a small residential population in Cambodia (Agusa et al. 2007), which was in agreement with a previous study among women with repeated miscarriages (Gerhard et al. 1998). A recent study of As exposure through well water consumption in Taiwan suggested that arsenic may impart an increased risk of erectile dysfunction through a reduction of circulating testosterone levels (Hsieh et al. 2008).
Although less attention has been given to the other metals in the past, a growing body of evidence suggests that certain essential or trace metals, including Cu, fluoride (F), Mn, Mo, and Se can also have adverse effects on male reproduction (Jeng et al. 2015; Yun Liu et al. 2019; Meeker et al. 2008; Telisman et al. 2000). In addition, most reports on the male reproductive effects of metals are from experimental animal, epidemiological, and occupational studies usually involving high doses not commonly encountered by children. Due to the widespread exposure of humans and known adverse effects related to essential and non-essential metal exposure, concern is growing that low-level exposure may also adversely affect reproductive developmental outcomes in boys. Moreover, only a few studies have investigated the cross-sectional relationships in boys, with the exception of two recent longitudinal studies in Russia and Mexico (Yun Liu et al. 2019; Williams et al. 2019), and none have examined exposure during in utero development and subsequent hormone levels during puberty, a time at which steroid hormones play an essential role in reproductive development (Cahill 2006; Parent et al. 2015; Sisk and Foster 2004; Spear 2000).
Therefore, the present study assessed whether in utero and prepubertal exposure to metals at relatively low doses altered reproductive hormone levels or timing and progression of sexual maturation in boys. We extended the limited metals studied on this topic and examined both essential and non-essential metals in relation to reproductive hormone levels and progression of sexual maturation in boys from ages 8–14 years to 10–18 years.
2.1. Study population
Participants in this study are part of the “Early Life Exposure in Mexico to Environmental Toxicants (ELEMENT)” project, a longitudinal cohort study of pregnant women in Mexico City and their children (Perng et al. 2019). Our analysis included women who were recruited from maternity hospitals during their first trimester between 1997 and 2004 and their children, as previously described (Ashrap et al. 2019; Lewis et al. 2013; Y. Liu et al. 2019; Perng et al. 2019; Wu et al. 2018). Mothers completed interview-based questionnaires at up to three prenatal visits [mean gestational age at visit 1: 13.5 (range:9–24) weeks, visit 2: 25.1 (range: 19–37) weeks, visit 3: 34.4 (range: 28–43) weeks] and provided spot urine samples at the third trimester visit. Between 2008 and 2011, a subset of their children (n = 250, 132 girls and 118 boys), who were then 8–14 years of age, were selected based on the availability of archived maternal biological specimens and re-contacted to participate in follow-up studies (i.e. early-teen visit). From those 250 children, 223 (89%) were enrolled again when they were age 10–18 years (late adolescence/adult) in a second follow-up study (i.e. late-teen visit), in 2013–2017 (Ashrap et al. 2019). Among those 223 children, 109 were boys (92% retention rate from early to late-teen visit) and the average follow up period was 3.5 years. Participants provided spot urine and fasting blood samples, anthropometry, and reported socio-demographic information via an interviewer-administered questionnaire. In the current analyses, we included male children who finished the early-teen visit (a majority of these same boys also completed late-teen visit) and had maternal urinary metal concentration measurements and/or their early-teen visit urinary metal measurements available (n = 118).
2.2. Metal Concentrations
Urinary metal concentrations of 14 metals and metalloids: aluminum (Al), As, barium (Ba), Cd, cobalt (Co), Cu, iron (Fe), Mn, Mo, nickel (Ni), Pb, antimony (Sb), selenium (Se), and Zn were measured in maternal third trimester urine and urine samples collected during the early-teen visit at age 8–14 years. Prenatal and peripubertal (early-teen) urine samples were collected in sterile cups, aliquoted within one hour after collection, frozen and stored at -80°F, and shipped on dry ice to McGill University (Montreal, Canada) for analysis. Urinary metals were measured using inductively coupled plasma mass spectrometry (ICPMS; Varian 820-MS, Inc., Palo Alto, California) as described previously (Basu et al. 2010; Srigboh et al. 2016). Accuracy and precision were measured using certified reference standards (Institut National de Santé Publique du Québec, or INSPQ) with coefficients of variation (CVs) ranging from 3 to 14%, and each batch run contained procedural blanks and replicate runs (Lewis et al. 2018; Srigboh et al. 2016). More details regarding quality control (QC) were previously described (Lewis et al. 2018). Values below the limit of detection (LOD) were replaced with the LOD/√2. Urinary specific gravity (SG) was measured using a handheld digital refractometer.
Pb exposure in this study was also measured in maternal patella/blood and early peripubertal blood, and results of these biomarkers in relation to pubertal development within this population have already been published (Jansen et al. 2018; Y. Liu et al. 2019). As patella and blood Pb levels are better biomarkers of long term Pb exposure, we excluded urinary Pb from the current analyses.
2.3. Hormones
Children provided fasting blood samples during the early-teen visit at age 8–14 years. Serum aliquots were separated and frozen at − 80 °C, and then sent to the Clinical Ligand Assay Service Satellite (CLASS) Laboratory at the University of Michigan (Ann Arbor, MI) for hormone analysis. Estradiol, testosterone, inhibin B, and sex hormone-binding globulin (SHBG) were measured in serum samples as biomarkers of puberty, and dehydroepiandrosterone sulfate (DHEA-S) was measured as a biomarker of adrenarche. Estradiol, total testosterone, SHBG, and DHEA-S were measured using an automated chemiluminescent immunoassay (Bayer Diagnostics ACS:180). Active inhibin B was assayed using Gen II ELISA (Beckman Coulter, Webster, TX). Values below the LOD were replaced with the LOD/√2.
2.4 Sexual Maturation
Two pediatricians evaluated male offspring for Tanner staging and testicular volume using standardized protocols during both follow-up visits at ages 8–14 and 10–18 years. To ensure consistency, pediatricians were trained prior to the start of each follow-up as previously described (Chavarro et al. 2017) to evaluate Tanner staging in male participants using standardized protocols. Genital stage (GD) was assessed as an indicator of puberty and pubic hair stage (PH) as an indicator of adrenarche, with stage 1 corresponding to no development and stage 5 corresponding to full development (Marshall and Tanner 1969). Right and left testicular volume (TV) were measured with an orchidometer, and the larger of the two measurements was used in analyses. As a testicular volume of 1–3 mL is considered prepubertal (Ankarberg-Lindgren and Norjavaara 2004; Mouritsen et al. 2013) and TV ≥ 20 mL was used as an indicator of sexual maturity (Ankarberg-Lindgren and Norjavaara 2004; Sergeyev et al. 2017), cutoffs of 3 mL and 20 mL were used to create a 3-level ordinal variable for testicular volume.
2.5 Covariates
Covariates in our analysis included: age at the early-teen visit, BMI z-scores at both visits, and household socioeconomic status at the late-teen visit. BMI z-scores were calculated based on the World Health Organization child reference curves for age and sex (WHO, 2007) for each follow up visit. A 7-level categorical variable for socioeconomic status (SES) was estimated using a validated scale consisting of thirteen questions on housing quality, services, material goods and head of household education (Asociación Mexicana de Agencias de Investigación de Mercados y Opinión Pública, AMAI version 13 × 6) (AMAI 2000; López 2008).
2.5. Statistical Methods
The geometric mean concentrations of each metal in urine samples from pregnant women and their children were calculated. The percent of urine samples with concentrations below the LOD were reported for each metal, and metals that were detected in less than 50% of samples were excluded from further analysis.
Multiple linear regressions were used to assess associations between urinary metal concentrations and peripubertal serum hormone concentrations, where serum hormones were natural log-transformed prior to analysis to achieve normal distribution. Metal concentrations from prenatal and peripubertal urine samples were entered into regression models separately and each model was adjusted for child age and BMI z-score, and for SG as a measure of urinary dilution. Results were calculated as the percent difference in hormone (95% confidence interval) per interquartile range (IQR) increase in urinary metal concentrations. Because reproductive hormones may differ greatly in boys at pre-pubertal stages (Tanner stage = 1) vs pubertal stages (Tanner stage > 1), we performed a secondary analysis in which we examined the associations between in utero and peripubertal exposures and reproductive hormone levels among subjects who were pre-pubertal (pubic hair Tanner stage = 1, n = 94).
Longitudinal analyses were conducted to explore the association between metals and early-teen and late-teen sexual maturation parameters using repeated measures generalized estimating equation (GEE). This allows us to take fuller advantage of the data that have been collected our longitudinal cohort study and use the additional within-person information to achieve increases in statistical power to detect associations (Sitlani et al. 2015). With separate models for each metal, the GEE approach was used to fit a multinomial (ordinal) regression model for pubertal stages at each visit as a function of metal exposure, age at early-teen visit and change in time, with adjustment for potential confounders:
g(E[Yij]) = β0 + β1Mij + β2Agei + β3Timeij + β4Mij*Timeij + β5Agei*Timeij (1)
g(E[Yij]) = β0 + β1Mij + β2Agei + β3Timeij + β4Mij*Timeij + β5Agei*Timeij
+β 6 BMIbase + β6 BMIvar (2)
Model (1) is the crude model where Y is an outcome of interest (Tanner stages/testicular volume), g is a link function (cumulative logit), i denotes subject number (1,…,n), and j denotes visit number (1, 2). M is ln-transformed metal exposure, A is the age at the early-teen visit, Time is the change in time between the early-teen and late-teen visit. Age at early-teen visit and change in time between two visits were included in the model to account for the effect of baseline age on attained Tanner stage or testicular volume, the natural pubertal progression across time, and the effect of baseline age on the natural pubertal progression. Model (2) is the final adjusted model, in which BMI z-score at the early-teen visit (BMIbase) and the change in BMI z-score from early-teen to late-teen visit (BMIvar) were included. The coefficients of interest are the cross-sectional effect of metal on tanner stage/testicular volume (β1) and the effect of metal on the progression of tanner stage/testicular volume over time (β4). Given the possibility that age and BMI z-score at the early-teen visit may be associated with Tanner stage and influence the future progression of Tanner stage, working independence was chosen as the covariance structure to ensure the validity of parameter estimates (Sullivan Pepe and Anderson 1994). We ran the GEE models both with and without BMI z-score (model 1 and 2) as BMI may be on the causal pathway between exposure and puberty. However, the magnitude of estimates from both models were almost identical, therefore we reported results from the models including BMI z-score. Results are presented as odds ratios (OR) and 95% confidence intervals (95% CI) per IQR increase in exposure. As few participants were categorized as having a PH Tanner stage = 3 and stage = 4 during the early-teen visit, the number of covariates we could reliably enter into models was limited (Peduzzi et al. 1996; Vittinghoff and McCulloch 2007). Thus, to minimize the number of covariates in GEE models, we included SG-corrected metal concentrations (rather than entering SG as a separate covariate which yielded similar results), using the following equation: Pc = P[(SGp – 1)/(SGi – 1)] where Pc is the SG corrected metal concentration (µg/L), P is the measured metal concentration, SGp is the median urinary specific gravity, and SGi is the individual’s urinary specific gravity.
Because SES could be a potential confounder, we ran a sensitivity analysis including SES as a covariate in models of hormones and maturation stages. Furthermore, as both low and high levels of essential metals are of concern, to explore potential non-linear associations we used adjusted generalized additive models (GAM) to graphically depict the cross-sectional relationship between metal concentrations and hormones and sexual maturation measurements. We also considered significance after adjusting for multiple testing using the Benjamini-Hochberg method (Benjamini and Hochberg 1995). Since Tanner stages/testicular volumes were not independent of each other, we calculated q values (adjusted p values) treating each outcome as a family of tests (11 tests per outcome). A cutoff of 0.15 for q value was used to further interpret main results with greater confidence. All analyses were performed using R version 3.5.2 and SAS 9.4.
3.1. Exposure and Outcome Distributions
The percent of samples with metal concentrations below the limit of detection, as well as the geometric means, standard deviations, and selected percentiles of sample concentrations from prenatal and peripubertal (early-teen) visits are shown in Table 1. Spearman correlations between the prenatal and peripubertal visit metal concentrations adjusted for SG are also presented in the table. With the exception of Sb and Fe, other metals were detected in > 50% of urine samples, therefore, Sb and Fe were excluded from further analysis. Weak correlations between maternal and peripubertal metal levels and weak to moderate correlations between different urinary metal concentrations within maternal and child samples have been previously reported elsewhere (Lewis et al. 2018). Distributions of Tanner stages of sexual maturation and testicular volume among the male children at the two follow up visits are reported (SI Table S1). At early -teen visit, the majority of the boys (n = 94, 81.7%) were at Tanner stage 1 for pubic hair development whereas 57 boys (49.6%) were at Tanner stage 1 for genital development. At the late-teen visit, boys moved to more advanced Tanner stages for sexual measurement- only 28 (26.4%) and 9 (7.5%) were at Tanner stage 1 for pubic hair development and genital development and 14 (13.2%) and 18 (13.2%) reached full development (Tanner stage = 5) for the two measurements. In terms of testicular volume distribution, the percentage of the boys in the prepubertal stage dropped from 14.8–0% from early-teen to late-teen visit.
| In utero | Peripubertal | |||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| LOD | %< LOD | GM | GSD | 25% | 50% | 75% | MAX | %< LOD | GM | GSD | 25% | 50% | 75% | MAX | P value b | |
| Essential metals | ||||||||||||||||
| Co | 0.4 | 0.0 | 1.2 | 1.9 | 0.8 | 1.2 | 2.1 | 5.2 | 0.0 | 0.7 | 1.5 | 0.6 | 0.8 | 0.9 | 1.6 | 0.63 |
| Cu | 48.2 | 46.3 | 86.1 | 4.4 | 34.1 | 50.2 | 71.9 | 2742 | 56.8 | 43.3 | 1.3 | 34.1 | 34.1 | 56.1 | 106 | 0.83 |
| Mn | 0.4 | 7.4 | 0.8 | 1.7 | 0.6 | 0.7 | 1.0 | 8.7 | 2.5 | 1.2 | 1.8 | 0.8 | 1.2 | 1.7 | 4.4 | 0.68 |
| Mo | 2.9 | 15.8 | 19.5 | 3.3 | 12.7 | 25.7 | 42.9 | 308 | 0.0 | 46.6 | 1.8 | 33.7 | 50.2 | 67.1 | 210 | 0.99 |
| Se | 10.5 | 3.2 | 29.9 | 1.7 | 23.0 | 32.6 | 43.1 | 120 | 1.7 | 47.2 | 1.7 | 36.3 | 53.0 | 65.6 | 141 | 0.26 |
| Zn | 0.1 | 1.2 | 271 | 2.2 | 160 | 298 | 451 | 1253 | 0.0 | 366 | 1.8 | 257 | 411 | 521 | 1200 | 0.08 |
| Non-essential Metals | ||||||||||||||||
| Al | 8.6 | 11.6 | 24.0 | 2.5 | 12.3 | 20.3 | 42.2 | 304 | 24.6 | 14.5 | 2.1 | 8.8 | 14.0 | 23.3 | 428 | 0.37 |
| As | 0.3 | 0.0 | 14.0 | 2.0 | 9.3 | 13.2 | 20.6 | 153 | 0.0 | 14.3 | 2.0 | 10.2 | 14.4 | 20.5 | 515 | 0.07 |
| Ba | 1.1 | 3.2 | 4.0 | 2.0 | 2.6 | 4.2 | 5.9 | 27.6 | 10.2 | 2.5 | 2.0 | 1.6 | 2.4 | 3.9 | 20.2 | 0.81 |
| Ni | 3.0 | 0.0 | 8.7 | 1.9 | 5.8 | 7.4 | 11.0 | 107 | 0.8 | 8.1 | 1.6 | 5.9 | 8.0 | 10.8 | 53.2 | 0.83 |
| Cd | 54.0 | 2.1 | 0.2 | 2.1 | 0.1 | 0.2 | 0.3 | 2.7 | 2.5 | 0.1 | 1.6 | 0.1 | 0.1 | 0.2 | 0.3 | 0.24 |
| a uncorrected for specific gravity | ||||||||||||||||
| b P value from Spearman correlation test between in utero and peripubertal metal concentration measurements. | ||||||||||||||||
| a Results are presented as the percent difference in peripubertal hormone levels associated with an interquartile range (IQR) increase in in utero and peripubertal metal | ||||||||||||||||
| b Linear regression models were adjusted for child age, BMI z-score and specific gravity | ||||||||||||||||
| * Significant associations with p value < 0.05 & q value < 0.15 | ||||||||||||||||
| * Significant associations with p value < 0.05 & q value > 0.15 | ||||||||||||||||
| a GEE models were adjusted for child age and BMI z-score (baseline and change) | ||||||||||||||||
| * Significant associations with p value < 0.05 & q value < 0.15 | ||||||||||||||||
| * Significant associations with p value < 0.05 & q value > 0.15 | ||||||||||||||||
| a GEE models are adjusted for child age and BMI z-score (baseline and change) | ||||||||||||||||
| * Significant associations with p value < 0.05 & q value < 0.15 | ||||||||||||||||
| * Significant associations with p value < 0.05 & q value > 0.15 | ||||||||||||||||
| Supplemental Information | ||||||||||||||||
| In Utero and Peripubertal Metal(loid)s Exposure in Relation to Reproductive Hormones and Sexual Maturation and Progression among boys in Mexico City | ||||||||||||||||
| Pahriya Ashrap, John D. Meeker, Brisa N. Sánchez, Niladri Basu, Marcela Tamayo-Ortiz, Maritsa Solano-González, Adriana Mercado-García, Martha M. Téllez-Rojo, Karen E. Peterson, Deborah J. Watkins* | ||||||||||||||||
| Table of Contents: | ||||||||||||||||
3.2 In utero and peripubertal metal exposure and peripubertal hormone levels
Associations between in utero and peripubertal metal concentrations and reproductive hormones are presented in Fig. 1 and SI Table S3. Positive associations were observed between some urinary essential metal concentrations and estradiol, testosterone, and SHBG. One IQR increase in in utero Zn concentration was associated with 13.7% higher serum estradiol levels (95% CI: 0.3, 28.8). In utero Co and Mn were positively associated with SHBG levels, with an IQR increase associated with 16% (95% CI:0.4, 34.2) and 14.2% (95%CI: 1.5, 28.5) higher serum SHBG after adjustment for child age, BMI z-score, and SG, respectively. As shown in Fig. 1, effect estimates for the association between both in utero essential and non-essential metal concentrations and testosterone were larger compared to other reproductive hormones, 51.3% for Mo, 35% for As, 38.9% for Cd. In models where reproductive hormones were regressed on concurrent peripubertal exposures, the strongest associations were observed also between metal concentrations and testosterone, particularly with non-essential metals Ni (%△/IQR: 35.1%, 95% CI: 2.6, 77.8) and Ba (%△/IQR: 59.1%, 95% CI: 22.5, 106.8). Peripubertal Ba concentration was also associated with higher estradiol (%△/IQR: 10.2%, 95% CI: 0.5, 20.9). However, no significant associations were detected between peripubertal metal exposures and DHEA-S or inhibin B.
After correcting for multiple testing, the associations of in utero Mo, As, and Cd with testosterone levels, as well as the association of peripubertal Ba with testosterone level had q-values < 0.15 (Fig. 1 and SI Table S3), providing greater confidence in these associations.
3.3 In utero and peripubertal metal exposure and sexual maturation
We have presented results from multiple ordinal regression models of in utero metal concentrations and Tanner stage and testicular volume in Fig. 2 and SI Table S4. Tanner stages and testicular volume at the early-teen visit were not associated with in utero metal concentrations, with the exception of a non-essential metal, Al; an IQR increase in in utero Al concentrations was associated with 3.6 times greater odds (95% CI: 1.67, 7.76) of being at a higher category of testicular volume versus lower categories. In the same figure and table, associations between in utero metal concentrations and pubertal development over time in boys are also presented. During the follow-up, an IQR increase in in utero concentrations of non-essential metalloid As was associated with 36% (OR/IQR: 0.64, 95% CI: 0.48, 0.85) lower odds of genital development progression per year, adjusting for age, BMI, and Tanner stage at the early-teen visit (GEE longitudinal model). In utero concentrations of non-essential metal(loid)s Al (OR/IQR: 0.61, 95% CI: 0.45, 0.83) and As (OR/IQR: 0.64, 95% CI: 0.43, 0.97) were associated with lower odds of progressing to a higher testicular volume category (i.e. 39 and 36% lower odds/IQR). Essential metal Zn was also associated with 38% lower odds of testicular volume progression (OR/IQR: 0.62, 95% CI: 0.44, 0.88).
Similarly, Fig. 3 and SI Table S4 show the cross-sectional and longitudinal associations between prepubertal metal concentrations and sexual maturation and progression. No significant associations were found between peripubertal urinary metal concentrations with Tanner stage for genital development or progression over follow-up, although several essential and non-essential metals were associated with pubic hair Tanner stages, testicular volume, and progressions. Peripubertal Zn concentration was associated with higher odds of being at a higher developmental stage for pubic hair (OR/IQR: 6.11, 95% CI: 1.89, 19.69) and testicular volume (OR/IQR: 5.39, 95% CI: 1.88, 15.49) at the early-teen visit, as well as slower progression of pubic hair development (OR/IQR: 0.47, 95% CI: 0.34, 0.67) and testicular volume (OR/IQR: 0.58, 95% CI: 0.40, 0.85) during the follow-up. Higher Mn was associated with 34% (OR/IQR: 0.66, 95% CI: 0.46, 0.96) lower pubic hair development progression only. Regarding non-essential metals, an IQR increase in peripubertal Ba concentration was associated with 2.3 times greater odds (95% CI: 1.15, 4.75) of being at higher Tanner stage for pubic hair development at age 8–14 years, but with 34% (OR/IQR: 0.66, 95% CI: 0.53, 0.83) lower odds of progressing to a higher pubic hair Tanner stage per year of follow-up. Higher peripubertal urinary Al was also associated with 47% (OR/IQR: 0.53, 95% CI: 0.40, 0.70) lower odds of pubic hair development progression per IQR increase.
All the associations described above had q-values < 0.15 (Fig. 2, Fig. 3, and SI Table S4) after correcting for multiple testing, except for the association between in utero As and testicular volume progressions. Output graphics produced from GAM models showed that there was no significant non-linear relationship between essential and non-essential metal concentrations and hormones and sexual maturation outcomes, after adjusting for the same sets of covariates.
3.4 Sensitivity Analysis
Results from the hormone subset analysis when we restricted to boys who were prepubertal (94 out of 118) at the early-teen visit are similar to the main analysis results (SI Table S5). For models regressing in utero metal concentrations, some effect estimates for DHEA_S, SHBG, and testosterone were no longer significant, potentially due to the small sample size. For models regressing peripubertal exposure, the notable effect estimates for testosterone were similar to those from the main analysis. The association between DHEA_S and Ni (%△/IQR = 20.2, 95%CI = 1.7, 42.0) and inhibin B and Al (%△/IQR =-19.2, 95%CI=-31.7, -4.4) became stronger and significant in the peripubertal subset analysis.
The magnitude of estimates from GEE models with and without BMI (SI Table S6) were almost identical. Findings from models adjusting for SES were generally consistent with metal and hormone associations observed in our main analyses; in SES-adjusted models, the association between in utero Co concentrations and higher SHBG (%△/IQR = 14.2, 95%CI=-2.9, 23.3) was slightly attenuated and no longer significant, while the association between peripubertal Al and inhibin B (%△/IQR=-14.1, 95%CI=-24.6, -2.2) was stronger and significant. In GEE models for Tanner stage or testicular volume status including SES finding remain consistent with the main models.
A high proportion of samples had metal concentrations above the LOD except for Cu, which was below the detection limit in 46% of samples. In a secondary analysis, we categorized urinary Cu concentrations into three groups. The low group consisted of values below the LOD, while the medium and high groups were made up of equalized bins among the detected values. We estimated the model parameters again and found that they were similar to the main parameter estimates.
Previous studies of metal exposure and male reproductive development have primarily been cross-sectional and focused on heavy metals, with the exception of “the Russian Children's Study” and “Early Life Exposure in Mexico to Environmental Toxicants (ELEMENT) Study” that assessed the longitudinal impact of blood Pb (Yun Liu et al. 2019; Williams et al. 2019). Thus, it is critical to evaluate the associations of essential and non-essential metals with reproductive hormones, sexual maturation, and progression among boys. Based on our longitudinal analysis of boys in a birth cohort in Mexico City who were examined at two peripubertal follow-up visits, we demonstrated a higher pubic hair development stage and testicular volume at the early-teen visit with higher non-essential metal concentrations, including in utero Al and peripubertal Ba and essential metal Zn concentration (peripubertal). We also found associations between in utero and peripubertal exposure to a number of both essential and non-essential metals and slowed progression of pubertal development across the follow-up period. In relation to hormone levels, higher non-essential metals, in utero As and Cd and peripubertal Ba concentrations as well as essential metal Mo measured in utero were associated with substantially higher testosterone levels. None of the associations between metals and hormones and measurements of sexual maturations appear to be non-linear.
To our knowledge, this is the first study to investigate the association between various in utero and peripubertal metals measured in urine and reproductive hormone levels in teenage boys. We reported a number of positive associations between specific metals measured in the urine and reproductive hormones, particularly testosterone. Some of our findings have not been reported elsewhere, such as relationships of essential minerals, e.g., Zn with estradiol or Co and Mn with SHBG. The positive association between relatively low Cd exposure and testosterone in this study is supported by epidemiological studies of non-occupational exposures among men (Jurasovic et al. 2004; Meeker et al. 2010; Menke et al. 2008; Telisman et al. 2007). However, in vivo and in vitro observations linked Cd with altered adrenal gland activity and induced oxidative stress, which could result in toxic effects on the testis and subsequently decrease in serum testosterone (Gay et al. 2013; Liu et al. 2009; Patra et al. 2011; Singhal et al. 1976; Siu et al. 2009).
Mo is an essential nutrient that is a cofactor for important enzymes involving in toxin removal (Schwarz 2016; Todd et al. 2017). While no prior studies have investigated the associations between Mo and hormones in boys, contrary to our finding on in utero Mo and increased testosterone, decreased testosterone levels and sperm concentrations were observed in males with increasing levels of Mo (Lewis and Meeker 2015; Meeker et al. 2008; Meeker et al. 2010). Several animal studies have also reported the reproductive toxicity of Mo, including declines in sperm concentration, motility and normal morphology, testicular degeneration, and reduced fertility (Jeter and Davis 1954; Pandey and Singh 2002; Thomas and Moss 1951; Vyskočil and Viau 1999). It is worth noting that the prenatal Mo levels were lower in our study population compared to the previous studies of Mo and reproductive outcomes and those reported in the National Health and Nutrition Examination Survey (NHANES) (Centers for Disease Control and Prevention 2018) (SI Table S2). It is not clear whether Mo levels measured in utero will have different effects on reproductive hormones measured during different life stages, which warrants further investigation. Although previous studies have not evaluated associations between Ba and reproductive hormones in human, our finding of positive associations between peripubertal Ba concentrations and serum testosterone and estradiol levels is consistent with previous studies among male zebrafish, where exposure to barium chloride significantly increased estradiol concentrations and transcripts of genes involved in the HPG axis (Kwon et al. 2016).
One of the main observations in this study is that Zn is associated with higher odds of pubic hair and testicular volume stage at the early-teen follow-up, but a slower progression of puberty from the early-teen to late-teen visit. Few cross-sectional studies on boys and girls also reported a significant positive relationship between serum or plasma zinc levels and stages of sexual maturation (Leonard et al. 1998; Onukwuli et al. 2018; Vivoli et al. 1990; Wagner et al. 1985), as well as reproductive hormones (testosterone). Zn is an essential trace metal and is fundamental for the development of the male reproductive system (Kerns et al. 2018; Mendoza et al. 2017). Studies on various animals described the role of Zn in advancing male puberty, through increased testicular activity, testosterone production, metabolism, and growth, primarily via activated the hypothalamus and the pituitary functions (Arangasamy et al. 2018; Dance et al. 2016; El-Masry et al. 1994; Fang and Furuhashi 1978; Geary et al. 2016; Underwood and Somers 1969). However, we did not observe a significant association between serum Zn and testosterone levels.
Pubic hair stages at the early-teen visit and progression were consistently associated with elevated concentrations of certain peripubertal metals, including As, Al, Mn, and Zn. The biological mechanisms that underlie the link between these essential and non-essential metals and pubertal development are uncertain but may be related to reproductive hormones. The main hormones responsible for pubic hair development in males are the androgens DHEA and testosterone (Breehl and Caban 2020; Rubinow and Schmidt 1996). In this cohort, the strongest observed associations were between in utero and peripubertal metal concentrations and increased testosterone, while no significant associations were seen in relation to DHEA-S. It is possible that these metals impact the appearance of pubic hair through changes in testosterone.
In this study, we observed slower progression of sexual maturation for those boys at higher Tanner stages at the early-teen visit which was also reported in studies explored the association between pubertal onset and progression (German et al. 2018; Llop-Viñolas et al. 2004; Pantsiotou et al. 2008). A compensatory mechanism similar to “catch-up growth” was proposed previously as a potential explanation for this observation (Ashrap et al. 2019; Wu et al. 2018). For those boys who had experienced a delayed pubertal development, their body systems may have responded by accelerating the tempo of pubertal progression (the change from lower stages of puberty to higher stages); while others who had experienced an advanced pubertal development may respond by slowing down the pubertal progression. This concept may explain the associations between metal exposure, earlier puberty onset at early-teen, and slower progression we found in this study. Future research is needed to establish this phenomenon and potential mechanisms.
The ELEMENT study, a prospective longitudinal birth cohort study in Mexico City, provided an opportunity to study the relationships of both in utero and peripubertal metal exposure on hormone levels and measures of sexual maturation and progression during the peripubertal period. Our results indicate that the in utero and peripubertal periods are vulnerable life stages, during which metal exposures may lead to disruption of male reproductive hormones and pubertal development. The findings also support that essential and non-essential metals have the potential to disrupt the onset and progression of puberty via interrupting the critical hormonal pathways. Several of the associations with hormones and sexual maturation are comparable to previous reports from human and animal research and supported by the current understanding of male pubertal development. However, there are inconsistent findings between the current study and previous studies and this may due to a number of reasons; 1) the exposure assessment approaches were different [sampling period, exposure matrices (i.e., blood, urine, hair)]. 2) most of the previous studies exploring the relationship between metals and hormones were conducted on adult population. 3) most studies examined the cross-sectional relationships whereas we examined the longitudinal association between metals and sexual maturation and progression. Limitations of our study include a somewhat small sample size and few observations for certain Tanner stages, which may result in imprecise effect estimates. Some of the metals measured in this study have a relatively short half-life in urine, so urinary levels at the time measurement may not fully characterize exposure during each specific window of development. Lastly, hormone levels were only measured at one time point and are likely to be subject to non-differential misclassification due to diurnal variation. Further research is warranted to prospectively explore the underlying mechanisms by which metals may affect male sexual maturation and progression in larger study populations.
Ethics approval and consent to participate
Research protocols were approved by the Institutional Review Board at University of Michigan, and the Research, Biosafety and Ethics in Research at the Mexico National Institute of Public Health. Prior to enrollment, informed consent from mothers and informed assent from offspring were obtained.
Consent for publication
Not applicable.
Availability of data and materials
All data generated and analyzed during this study are not publicly available due to the Institutional Review Board restrictions.
Competing interests
The authors declare that they have no competing interests.
Funding
This work was supported by U.S. Environmental Protection Agency (US EPA) grants RD834800 and RD83543601 and National Institute for Environmental Health Sciences (NIEHS) grants P20 ES018171, P01 ES02284401, and P30 ES017885. Its contents are solely the responsibility of the grantee and do not necessarily represent the official views of the US EPA. Further, the US EPA does not endorse the purchase of any commercial products or services mentioned in the publication. This work was also supported and partially funded by the National Institute of Public Health, Ministry of Health of Mexico.
Authors’ Contributions
PA conducted data analyses and drafted the manuscript. JDM, KEP, MTR and DJW conceived the study and oversaw its coordination. NB helped oversee the study, data acquisition, metals analysis, and advised the interpretation and drafting of the manuscript. BNS helped oversee the study and advised during the data analysis. MSG and AMG participated in the collection and assembly of data. All authors read and approved the final manuscript.
Acknowledgments
The authors acknowledge the research staff at participating hospitals and the American British Cowdray Hospital in Mexico City for providing research facilities. We thank the mothers and children for participating in the study.
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