Urinary phthalate metabolite concentrations during four windows spanning puberty (prepuberty through sexual maturity) and association with semen quality among young Russian men.

AIM: To prospectively investigate the associations of urinary phthalate metabolite concentrations measured at four time points spanning pubertal development with semen parameters in Russian men. DESIGN: 516 boys were enrolled at ages 8-9 years (2003-2005) and followed annually. METHODS: Urine samples were collected annually and pooled into four exposure windows [prepuberty, early puberty, late puberty and sexual maturity] based on physician assessed Tanner genitalia stages and testicular volume. Fifteen phthalate metabolites were quantified using isotope dilution HPLC-MS/MS at Moscow State University. We calculated molar sums (∑) of di-2-ethylhexyl phthalate (DEHP), di-isononyl phthalate (DiNP), di-isodecyl phthalate (DiDP) and anti-androgenic phthalate (AAP) metabolites. At sexual maturity (ages 18-19 years), the men provided 1-2 semen samples for analysis. We estimated the associations of quintiles of urinary ∑phthalate metabolites as well as mono-butyl phthalate (MnBP), mono-isobutyl phthalate (MiBP), and mono-benzyl phthalate (MBzP) at each pubertal window, with semen parameters by fitting generalized linear mixed models with random intercepts and adjusting for confounders. RESULTS: A total of 223 men who provided semen samples had phthalates measured at one or more pubertal windows. Higher urinary concentrations of ∑DiNP metabolites during late puberty were related to poorer semen quality (men with the highest quintile of urinary ∑DiNP had 30% lower sperm concentration, 32% lower count and 30% lower progressive motile count, compared to men in the lowest quintile). Also, young men with higher urinary concentrations of MiBP metabolites in early puberty tended to have poorer semen quality. No associations were observed for ∑DEHP metabolites, ∑DiDP metabolites, ∑AAP, MBzP or MnBP metabolites with semen quality parameters. CONCLUSIONS: ∑DiNP metabolites measured during late puberty and MiBP metabolites at early puberty were related to poorer semen quality, highlighting the importance of considering specific windows of exposure when investigating chemical exposures in relation to measures of reproductive health in men.

Association of peripubertal blood lead levels with reproductive hormones and semen parameters in a longitudinal cohort of Russian men.

STUDY QUESTION: Are peripubertal blood lead levels (BLLs) associated with semen parameters and serum reproductive hormones among young Russian men? SUMMARY ANSWER: We observed a suggestion of lower ejaculate volume with higher peripubertal BLL but no associations of BLLs with reproductive hormones measured throughout adolescence or with other sperm parameters measured at adulthood. WHAT IS KNOWN ALREADY: Lead is a known reproductive toxicant and endocrine disruptor. Previous literature has shown associations between high lead exposure and poorer semen quality both in occupationally and environmentally exposed men. However, to our knowledge, no longitudinal studies have explored the association of childhood lead exposure with semen parameters and reproductive hormones in young men. STUDY DESIGN, SIZE, DURATION: The Russian Children's Study is a prospective cohort study that enrolled 516 boys at age 8-9 years in 2003-2005 and followed them annually for 10 years. BLLs were measured at entry and lifestyle and health questionnaires were completed. Reproductive hormones were measured in blood samples collected every 2 years. PARTICIPANTS/MATERIALS, SETTING, METHODS: Among the 516 boys enrolled, 481 had BLLs measured at entry. Of these, 453 had at least one measurement of serum testosterone, follicle stimulating hormone (FSH) or luteinizing hormone (LH) (median = 5 samples per boy) and 223 had semen samples collected ∼10 years after enrolment. Semen assessment included ejaculated volume, sperm concentration, progressive motility and total sperm count, and parameters were categorized using published andrology standards for low semen quality based on sperm count and motility. Linear mixed models were used to examine the associations of log-transformed BLLs (and BLL categories) with reproductive hormones and semen parameters, adjusting for potential confounders. MAIN RESULTS AND THE ROLE OF CHANCE: Among the 223 young men with peripubertal BLLs and at least one semen sample (total samples = 438), the median (interquartile range) BLL was 3 (2, 5) µg/dl and 27% had BLL ≥5 µg/dl. Overall, 49% of the semen samples fell below reference levels for sperm count and/or motility. Men with peripubertal BLL ≥5 µg/dl had significantly lower ejaculated volume than those with BLL <5 µg/dl (mean = 2.42 vs 2.89 ml, P = 0.02), but this difference was attenuated in adjusted models (mean = 2.60 vs 2.83 ml, P = 0.25). No associations were observed between BLL measured at age 8-9 years and reproductive hormone levels or sperm parameters, including sperm concentration, total count, progressive motility and total progressive motile sperm count, or with the probability of having low semen quality based on sperm count/motility. LIMITATIONS, REASONS FOR CAUTION: Only a subset of the original cohort participated in the semen quality portion of the study, although inverse probability weighting was used to account for possible selection bias. BLLs were only measured at a single time in peripuberty, and other exposure time periods, including later or longer-term childhood exposure, may be more predictive of semen quality. The young men were also exposed to other chemical contaminants before and during pubertal development. WIDER IMPLICATIONS OF THE FINDINGS: While semen volume often receives less attention than other sperm parameters, it is an important component of male fertility. Additional prospective studies covering different exposure windows and including other seminal plasma biomarkers are warranted to explore our finding of potentially lower ejaculated volume with higher BLLs and to confirm the lack of associations for other semen parameters among youth exposed to environmental BLLs. STUDY FUNDING/COMPETING INTEREST(S): Funding was provided through grants R01ES0014370 and P30ES000002 from the National Institute of Environmental Health Sciences, grant R82943701 from the U.S. Environmental Protection Agency, and grant 18-15-00202 from the Russian Science Foundation (O.S and Y.D.). All authors report no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Optimization of small RNA extraction and comparative study of NGS library preparation from low count sperm samples.

Recent studies demonstrate that sperm epigenome is a vehicle that conveys paternal experiences to offspring phenotype. That evidence triggers interest of both experimental and epidemiological studies of epigenetic markers in sperm. Since samples are often unique in epidemiological studies, a careful and efficient use of the material is a critical requirement. The goal of this study was to provide optimization of methods for the isolation of small RNAs from spermatozoa and library preparation for sequencing. A total 67 fractionated sperm samples from the Russian Children's Study biobank prospectively collected at 18-20 years of age were used to isolate small RNAs with median (IQR) input total sperm count 17.0 (7.4-35.9) million. Twenty-four pairs of libraries were prepared using the NEBNext and NEXTFlex kits, 19 libraries using NEBNext and 6 using NEXTFlex. All libraries were sequenced on NextSeq 500, and the results were evaluated as a function of the number of small non-coding RNA (sncRNA) detected, quality parameters of sequencing libraries, as well as technical features of sample preparation. Although the same amount of miRNA input was used for NEBNext and NEXTFlex libraries, the concentration of DNA in NEBNext libraries was significantly higher in comparison with NEXTFlex libraries. In high input (sperm count >28 million and more than 25 ng miRNA in library) NEXTFlex Small RNA-Seq kit detected more microRNAs. In low input, the NEBNext proved more effective. The tricks and traps to protocol optimization are presented, including an efficient and effector gel-based system for the removal of sequencing library adaptors.

Peripubertal serum concentrations of organochlorine pesticides and semen parameters in Russian young men.

BACKGROUND: Epidemiologic literature on the relation of organochlorine pesticides (OCPs) with semen quality among adult men has been inconclusive, and no studies have prospectively explored the association between peripubertal serum OCPs and semen parameters in young men. OBJECTIVE: To evaluate prospective associations of peripubertal serum concentrations of hexachlorobenzene (HCB), ß-hexachlorocylohexane (ß-HCH), and p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) with semen parameters among young Russian men. METHODS: This prospective cohort study included 152 young men who enrolled in the Russian Children's Study (2003-2005) at age 8-9 years and were followed annually until young adulthood. HCB, ß-HCH, and p,p'-DDE concentrations were measured at the CDC by mass spectrometry in serum collected at enrollment. Between 18 and 23 years, semen samples (n = 298) were provided for analysis of volume, concentration, and progressive motility; we also calculated total sperm count and total progressive motile count. Linear mixed models were used to examine the longitudinal associations of quartiles of serum HCB, ß-HCH and p,p'-DDE with semen parameters, adjusting for total serum lipids, body mass index, smoking, abstinence time and baseline dietary macronutrient intake. RESULTS: Lipid-adjusted medians (IQR) for serum HCB, ßHCH and p,p-DDE, respectively, were 150 ng/g lipid (102-243), 172 ng/g lipid (120-257) and 275 ng/g lipid (190-465). In adjusted models, we observed lower ejaculated volume with higher serum concentrations of HCB and ßHCH, along with reduced progressive motility with higher concentrations of ßHCH andp,p-DDE. Men in the highest quartile of serum HCB had a mean (95% Confidence Interval, CI) ejaculated volume of 2.25 mL (1.89, 2.60), as compared to those in the lowest quartile with a mean (95% CI) of 2.97 mL (2.46, 3.49) (p = 0.03). Also, men in the highest quartile of serum p,p-DDE had a mean (95% CI) progressive motility of 51.1% (48.6, 53.7), as compared to those in the lowest quartile with a mean (95% CI) of 55.1% (51.7, 58.5) (p = 0.07). CONCLUSION: In this longitudinal Russian cohort study, peripubertal serum concentrations of selected OCPs were associated with lower ejaculated volume and progressive motility highlighting the importance of the peripubertal window when evaluating chemical exposures in relation to semen quality.

Peripubertal serum dioxin concentrations and subsequent sperm methylome profiles of young Russian adults.

BACKGROUND: The association of exposure to endocrine disrupting chemicals in the peripubertal period with subsequent sperm DNA methylation is unknown. OBJECTIVE: We examined the association of peripubertal serum 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) concentrations with whole-genome bisulfite sequencing (WGBS) of sperm collected in young adulthood. METHODS: The Russian Children's Study is a prospective cohort of 516 boys who were enrolled at 8-9 years of age and provided semen samples at 18-19 years of age. WGBS of sperm was conducted to identify differentially methylated regions (DMR) between highest (n = 4) and lowest (n = 4) peripubertal TCDD groups. RESULTS: We found 52 DMRs that distinguished lowest and highest peripubertal serum TCDD concentrations. One of the top scoring networks, "Cellular Assembly and Organization, Cellular Function and Maintenance, Carbohydrate Metabolism", identified estrogen receptor alpha as its central regulator. CONCLUSION: Findings from our limited sample size suggest that peripubertal environmental exposures are associated with sperm DNA methylation in young adults.

A Longitudinal Study of Peripubertal Serum Organochlorine Concentrations and Semen Parameters in Young Men: The Russian Children's Study.

BACKGROUND: Exposures to endocrine-disrupting chemicals during critical phases of testicular development may be related to poorer semen parameters. However, few studies have assessed the association between childhood organochlorine (OC) exposure and adult semen parameters. OBJECTIVE: We examined whether peripubertal serum OC concentrations are associated with semen parameters among young Russian men. METHODS: From 2003 through 2005, 516 boys were enrolled at age 8-9 years and followed for up to 10 years. Serum OCs were measured in the enrollment samples using high-resolution mass spectrometry. At 18-19 years, 133 young men provided 1 or 2 semen samples (256 samples) collected approximately 1 week apart, which were analyzed for volume, sperm concentration, and motility. Unadjusted and adjusted linear mixed models were used to examine the associations of quartiles of lipid-standardized concentrations of dioxins [2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorinated dibenzo-p-dioxins (PCDDs)], furans, polychlorinated biphenyls (PCBs), and corresponding toxic equivalents (TEQs) with semen parameters. RESULTS: The median (range) for TCDD was 2.9 (0.4-12.1) pg/g lipid and PCDD TEQ was 8.7 (1.0-36.0) pg TEQ/g lipid. Higher quartiles of TCDD and PCDD TEQs were associated with lower sperm concentration, total sperm count, and total motile sperm count (p-trends ≤ 0.05). The highest quartile of peripubertal serum TCDD concentrations was associated with a decrease (95% CI) of 40% (18, 66%), 29% (3, 64%), and 30% (2, 70%) in sperm concentration, total sperm count, and total motile sperm count, respectively, compared with the lowest quartile. Similar associations were observed for serum PCDD TEQs with semen parameters. Serum PCBs, furans, and total TEQs were not associated with semen parameters. CONCLUSION: Higher peripubertal serum TCDD concentrations and PCDD TEQs were associated with poorer semen parameters. Citation: Mínguez-Alarcón L, Sergeyev O, Burns JS, Williams PL, Lee MM, Korrick SA, Smigulina L, Revich B, Hauser R. 2017. A longitudinal study of peripubertal serum organochlorine concentrations and semen parameters in young men: the Russian Children's Study. Environ Health Perspect 125:460-466; http://dx.doi.org/10.1289/EHP25.