Haotian Wu

Environmental Susceptibility of the Sperm Epigenome During Windows of Male Germ Cell Development

Male germ cells require multiple epigenetic reprogramming events during their lifespan to achieve reproductive capacity. An emerging body of compelling data demonstrates that environmental exposures can be embodied within the developing male germ cell as epigenetic marks. In turn, these epigenetic marks can impart information at fertilization to affect the trajectory of offspring health and development. While it is recognized that in utero epigenetic reprogramming of male germ cells is a particularly susceptible window to environmental exposures, other such windows exist during germ cell development. The objective of this review is to discuss epigenetic reprogramming events during male germ cell development and to provide supporting evidence from animal and human studies that during specific periods of development, germ cells are susceptible to environmentally induced epigenetic errors. Moving forward, the nascent field of sperm epigenetics research is likely to advance our understanding of paternal environmental determinants of offspring health and development.

Coffee, tea, and melanoma risk among postmenopausal women

Laboratory research suggests that components in coffee and tea may have anticarcinogenic effects. Some epidemiologic studies have reported that women who consume coffee and tea have a lower risk for melanoma. We assessed coffee, tea, and melanoma risk prospectively in the Women’s Health Initiative – Observational Study cohort of 66 484 postmenopausal women, followed for an average of 7.7 years. Coffee and tea intakes were measured through self-administered questionnaires at baseline and at year 3 of follow-up. Self-reported incident melanomas were adjudicated using medical records. Cox proportional hazard models were used to estimate risk, adjusting for covariates, with person-time accumulation until melanoma diagnosis (n=398), death, loss to follow-up, or through 2005. Daily coffee [hazard ratio (HR)=0.87, 95% confidence interval (CI) 0.68–1.12] and tea (HR=1.03, 95% CI 0.81–1.31) intakes were not significantly associated with melanoma risk compared with nondaily intake of each beverage. No significant trends were observed between melanoma risk and increasing intakes of coffee (P for trend=0.38) or tea (P for trend=0.22). Women who reported daily coffee intake at both baseline and year 3 had a significantly decreased risk compared with women who reported nondaily intake at both time points (HR=0.68, 95% CI 0.48–0.97). Consistent daily tea intake was not associated with decreased melanoma risk. Overall, there is no strong evidence that increasing coffee or tea consumption can lead to a lower melanoma risk. We observed a decrease in melanoma risk among long-term coffee drinkers, but the lack of consistency in the results by dose and type cautioned against overinterpretation of the results.

Preconception urinary phthalate concentrations and sperm DNA methylation profiles among men undergoing IVF treatment: a cross-sectional study

STUDY QUESTION Are preconception phthalate and phthalate replacements associated with sperm differentially methylated regions (DMRs) among men undergoing IVF? SUMMARY ANSWER Ten phthalate metabolites were associated with 131 sperm DMRs that were enriched in genes related to growth and development, cell movement and cytoskeleton structure. WHAT IS KNOWN ALREADY Several phthalate compounds and their metabolites are known endocrine disrupting compounds and are pervasive environmental contaminants. Rodent studies report that prenatal phthalate exposures induce sperm DMRs, but the influence of preconception phthalate exposure on sperm DNA methylation in humans is unknown. STUDY DESIGN, SIZE, DURATION An exploratory cross-sectional study with 48 male participants from the Sperm Environmental Epigenetics and Development Study (SEEDS). PARTICIPANTS/MATERIALS, SETTING, METHODS The first 48 couples provided a spot urine sample on the same day as semen sample procurement. Sperm DNA methylation was assessed with the HumanMethylation 450 K array. Seventeen urinary phthalate and 1,2-Cyclohexane dicarboxylic acid diisononyl ester (DINCH) metabolite concentrations were measured from spot urine samples. The A-clust algorithm was employed to identify co-regulated regions. DMRs associated with urinary metabolite concentrations were identified via linear models, corrected for false discovery rate (FDR). MAIN RESULTS AND ROLE OF CHANCE Adjusting for age, BMI, and current smoking, 131 DMRs were associated with at least one urinary metabolite. Most sperm DMRs were associated with anti-androgenic metabolites, including mono(2-ethylhexyl) phthalate (MEHP, n = 83), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP, n = 16), mono-n-butyl phthalate (MBP, n = 22) and cyclohexane-1,2-dicarboxylic acid-monocarboxy isooctyl (MCOCH, n = 7). The DMRs were enriched in lincRNAs as well as in regions near coding regions. Functional analyses of DMRs revealed enrichment of genes related to growth and development as well as cellular function and maintenance. Finally, 13% of sperm DMRs were inversely associated with high quality blastocyst-stage embryos after IVF. LIMITATIONS, REASONS FOR CAUTION Our modest sample size only included 48 males and additional larger studies are necessary to confirm our observed results. Non-differential misclassification of exposure is also a concern given the single spot urine collection. WIDER IMPLICATIONS OF THE FINDINGS To our knowledge, this is the first study to report that preconception urinary phthalate metabolite concentrations are associated with sperm DNA methylation in humans. These results suggest that paternal adult environmental conditions may influence epigenetic reprogramming during spermatogenesis, and in turn, influence early-life development. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by grant K22-ES023085 from the National Institute of Environmental Health Sciences. The authors declare no competing interests.

Rapid method for the isolation of mammalian sperm DNA

The unique DNA packaging of spermatozoa renders them resistant to DNA isolation techniques used for somatic cells, requiring alternative methods that are slow and labor intensive. Here we present a rapid method for isolating high-quality sperm DNA. Isolated human sperm cells were homogenized with 0.2 mm steel beads for 5 min at room temperature in the presence of guanidine thiocyanate lysis buffer supplemented with 50 mM tris(2-carboxyethyl)phosphine (TCEP). Our method yielded >90% high-quality DNA using 3 different commercially available silica-based spin columns. DNA yields did not differ between immediate isolation (2.84 ± 0.04 pg/cell) and isolation after 2 weeks of homogenate storage at room temperature (2.91 ± 0.13 pg/cell). DNA methylation analyses revealed similar methylation levels at both time points for three imprinted loci. Our protocol has many advantages: it is conducted at room temperature; lengthy proteinase K (ProK) digestions are eliminated; the reducing agent, TCEP, is odorless and stable at room temperature; nucleic acids are stabilized, allowing storage of homogenate; and it is adaptable for other mammalian species. Taken together, the benefits of our improved method have important implications for settings where sample processing constraints exist.

Parental contributions to early embryo development: influences of urinary phthalate and phthalate alternatives among couples undergoing IVF treatment

STUDY QUESTION Are preconception urinary concentrations of phthalates and phthalate alternatives associated with diminished early stage embryo quality in couples undergoing IVF? SUMMARY ANSWER Male, but not female, urinary concentrations of select metabolites of phthalates and phthalate alternatives are associated with diminished blastocyst quality. WHAT IS KNOWN ALREADY Although phthalates are endocrine disrupting compounds associated with adverse reproductive health, they are in widespread use across the world. Male and female preconception exposures to select phthalates have been previously associated with adverse reproductive outcomes in both the general population and in those undergoing IVF. STUDY DESIGN, SIZE, DURATION This prospective cohort included 50 subfertile couples undergoing IVF in western Massachusetts. PARTICIPANTS/MATERIALS, SETTING, METHODS This study includes the first 50 couples recruited from the Baystate Medical Centers Fertility Center in Springfield, MA, as part of the Sperm Environmental Epigenetics and Development Study (SEEDS). Relevant data from both partners, including embryo quality at the cleavage (Day 3) and blastocyst (Day 5) stages, were collected by clinic personnel during the normal course of an IVF cycle. A spot urine sample was collected from both male and female partners on the same day as semen sample procurement and oocyte retrieval. Concentrations of 17 urinary metabolite were quantified by liquid chromatography mass spectrometry and normalized via specific gravity. Generalized estimating equations were used to estimate odds ratios (OR) and 95% CI, with urinary phthalates and phthalate alternatives fitted as continuous variables and embryo quality as a binary variable. MAIN RESULTS AND THE ROLE OF CHANCE The 50 couples contributed 761 oocytes, of which 423 progressed to the cleavage stage, 261 were high-quality cleavage stage embryos, 137 were transferrable quality blastocysts and 47 were high-quality blastocysts. At the cleavage stage, male urinary monoethyl phthalate concentrations were positively associated with high-quality cleavage stage embryos (OR = 1.20, 95% CI 1.01–1.43, P = 0.04); no other significant associations were observed at this stage. At the blastocyst stage, male urinary concentrations of monobenzyl phthalate (OR = 0.55, 95% CI 0.36–0.84, P = 0.01), mono-3-hydroxybutyl phthalate (OR = 0.37, 95% CI 0.18–0.76, P = 0.01), mono-n-butyl phthalate (OR = 0.55, 95% CI 0.42–0.73, P < 0.01) and monomethyl phthalate (OR = 0.39, 95% CI 0.26–0.60, P < 0.01) were inversely associated with high-quality blastocysts. A borderline statistically significant relationship was observed for male concentrations of mono(2-ethylhexyl) phthalate (OR = 0.52, 95% CI 0.27–1.00, P = 0.05) and cyclohexane-1,2-dicarboxylic acid-monocarboxy isooctyl ester (OR = 0.21, 95% CI 0.04–1.03, P = 0.05) at the blastocyst stage. Similar inverse associations were observed between male urinary phthalate metabolite concentrations and likelihood of transferrable quality blastocysts. For female partners, select metabolites were positively associated with odds of high or transferrable blastocyst quality, but the observed associations were not consistent across blastocyst quality measures or between sex-specific and couples-level models. All models were adjusted for age of both partners, urinary metabolite concentrations of female partners and male infertility status, while models of blastocysts were additionally adjusted for embryo quality at cleavage stage. LIMITATIONS, REASONS FOR CAUTION Our modest sample included only 50 couples contributing one cycle each. In addition, non-differential misclassification of exposure remains a concern given the single-spot urine collection and the short half-life of phthalates. WIDER IMPLICATIONS OF THE FINDINGS Our results suggest an inverse association between male preconception concentrations of select phthalate metabolites and blastocyst quality, likely occurring after genomic activation. If corroborated with other studies, such findings will have public health and clinical significance for both the general population and those undergoing IVF. STUDY FUNDING/COMPETING INTERESTS This work was generously supported by grant K22-ES023085 from the National Institute of Environmental Health Sciences. The authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A.

Urinary phthalate and phthalate alternative metabolites and isoprostane among couples undergoing fertility treatment

Background: Epidemiological data suggest associations between phthalate exposures to a variety of adverse reproductive outcomes including reduced sperm quality and reproductive success. While mechanisms of these associations are not fully elucidated, oxidative stress has been implicated as a potential mediator. We examined associations of urinary metabolites of phthalates and phthalate alternative plasticizers with oxidative stress among couples seeking fertility treatment. Methods: Seventeen urinary plasticizer metabolites and 15‐F2t isoprostane, a biomarker of oxidative stress, were quantified in spot samples from 50 couples seeking fertility treatment who enrolled in the Sperm Environmental Epigenetics and Development Study during 2014–2015. Results: In multivariable analyses, percent change in isoprostane was positively associated with interquartile range increases for the oxidative metabolites of di‐2‐ethylhexyl phthalate, [mono‐2‐ethyl‐5‐hydroxyhexyl phthalate (MEHHP; 20.0%, p=0.02), mono‐2‐ethyl‐5‐oxohexyl phthalate (MEOHP; 24.1%, p=0.01), and mono‐2‐ethyl‐5‐carboxypentyl phthalate (MECPP; 24.1%, p=0.004)], mono‐isobutyl phthalate (MiBP; 17.8%, p=0.02), mono‐hydroxyisobutyl phthalate (MHiBP; 27.5%, p=0.003), and cyclohexane‐1,2‐dicarboxylic acid mono‐hydroxy‐isononyl ester (MHINCH; 32.3%, p=0.002). Stratification of participants by sex revealed that isoprostane was positively associated with MHiBP (41.4%, p=0.01) and monocarboxy‐isononyl phthalate (MCNP; 26.0%, p=0.02) among females and MEOHP (35.8%, p=0.03), MiBP (29.2%, p=0.01), MHiBP (34.7%, p=0.007) and MHINCH (49.0%, p=0.002) among males. Conclusions: Our results suggest that exposure to phthalates and phthalate replacements are associated with higher levels of oxidative stress in a sex‐specific manner. Additional studies are needed to replicate our findings and to examine the potential health implications of the use of phthalates and alternative phthalates in consumer end products. HighlightsPhthalates were positively associated with isoprostane in an IVF population.The observed associations were sex‐specific.Phthalate replacements were associated with isoprostane in males.While limited by sample size, findings are concordant with previous reports.

Associations of urinary phthalate metabolites and lipid peroxidation with sperm mitochondrial DNA copy number and deletions

Background Phthalates, a chemical class of plasticizers, are ubiquitous environmental contaminants that have been associated with oxidative stress. Mitochondria DNA copy number (mtDNAcn) and DNA deletions (mtDNAdel) are emerging biomarkers for cellular oxidative stress and environment exposures. Objectives To examine associations of urinary phthalate metabolite and isoprostane concentrations on sperm mtDNAcn and mtDNAdel in male partners undergoing assisted reproductive technologies (ART). Methods Ninety‐nine sperm samples were collected from male partners undergoing ART at Baystate Medical Center in Springfield, MA as part of the Sperm Environmental Epigenetics and Development Study (SEEDS). Seventeen urinary phthalate metabolite concentrations were analyzed by the Centers for Disease Control using tandem mass spectrometry. Urinary 15‐F2t‐isoprostane concentrations, a biomarker of lipid peroxidation, were measured using a competitive enzyme‐linked immunosorbent assay. A triplex qPCR method was used to determine the relative quantification of mtDNAcn and mtDNAdel. Results Sperm mtDNAcn and mtDNAdel were positively correlated (Spearman rho = 0.31; p = .002). Adjusting for age, BMI, current smoking, race, and measurement batch, urinary monocarboxy‐isononyl phthalate (MCNP) concentrations were positively associated with mtDNAcn (&bgr; = 1.63, 95% CI: 0.14, 3.11). Other urinary phthalate metabolite and isoprostane concentrations were not associated with sperm mtDNAcn or mtDNAdel. Conclusions Among this cohort of male ART participants, those with higher MCNP had higher mtDNAcn; other phthalate metabolites and isoprostane were not associated with mtDNAcn and mtDNAdel. Given our relatively small sample size, our results should be interpreted with caution. Future research is needed to replicate the findings in larger studies and among sperm samples obtained from the general population. HighlightsSperm mitochondria DNA copy number (mtDNAcn) and deletions (mtDNAdel) were positively associated.Urinary monocarboxy‐isononyl phthalate (MCNP) concentrations were positively associated with mtDNAcn.Urinary phthalate metabolite concentrations were not associated with sperm mtDNAdel.Urinary isoprostane concentrations were not associated with sperm mtDNAcn or mtDNAdel.

Relationships between Global DNA Methylation in Circulating White Blood Cells and Breast Cancer Risk Factors

It is not yet clear whether white blood cell DNA global methylation is associated with breast cancer risk. In this review we examine the relationships between multiple breast cancer risk factors and three markers of global DNA methylation: LINE-1, 5-mdC, and Alu. A literature search was conducted using Pubmed up to April 1, 2016, using combinations of relevant outcomes such as “WBC methylation,” “blood methylation,” “blood LINE-1 methylation,” and a comprehensive list of known and suspected breast cancer risk factors. Overall, the vast majority of reports in the literature have focused on LINE-1. There was reasonably consistent evidence across the studies examined that males have higher levels of LINE-1 methylation in WBC DNA than females. None of the other demographic, lifestyle, dietary, or health condition risk factors were consistently associated with LINE-1 DNA methylation across studies. With the possible exception of sex, there was also little evidence that the wide range of breast cancer risk factors we examined were associated with either of the other two global DNA methylation markers: 5-mdC and Alu. One possible implication of the observed lack of association between global WBC DNA methylation and known breast cancer risk factors is that the association between global WBC DNA methylation and breast cancer, if it exists, is due to a disease effect.

White blood cell DNA methylation and risk of breast cancer in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO)

BackgroundSeveral studies have suggested that global DNA methylation in circulating white blood cells (WBC) is associated with breast cancer risk.MethodsTo address conflicting results and concerns that the findings for WBC DNA methylation in some prior studies may reflect disease effects, we evaluated the relationship between global levels of WBC DNA methylation in white blood cells and breast cancer risk in a case-control study nested within the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial (PLCO) cohort. A total of 428 invasive breast cancer cases and 419 controls, frequency matched on age at entry (55–59, 60–64, 65–69, ≥70 years), year of entry (on/before September 30, 1997, on/after October 1, 1997) and period of DNA extraction (previously extracted, newly extracted) were included. The ratio of 5-methyl-2’ deoxycytidine [5-mdC] to 2’-deoxyguanine [dG], assuming [dG] = [5-mdC] + [2’-deoxycytidine [dC]] (%5-mdC), was determined by liquid chromatography-electrospray ionization-tandem mass spectrometry, an especially accurate method for assessing total genomic DNA methylation.ResultsOdds ratio (OR) estimates and 95% confidence intervals (CI) for breast cancer risk adjusted for age at entry, year of entry, and period of DNA extraction, were 1.0 (referent), 0.89 (95% CI, 0.6–1.3), 0.88 (95% CI, 0.6–1.3), and 0.84 (95% CI, 0.6–1.2) for women in the highest compared to lowest quartile levels of %5md-C (p for trend = .39). Effects did not meaningfully vary by time elapsed from WBC collection to diagnosis.DiscussionThese results do not support the hypothesis that global DNA hypomethylation in WBC DNA is associated with increased breast cancer risk prior to the appearance of clinical disease.