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Background: Phthalates have been linked to adverse male reproductive health, including poor sperm quality and embryo quality as well as a longer time to pregnancy (months of unprotected intercourse before conception occurs). The present study aimed to evaluate the effect of preconception exposure to two ubiquitous phthalate chemicals, di(2‐ethylhexyl) phthalate (DEHP), di‐n‐butyl phthalate (DBP), and their mixture on sperm function, fertilization, and embryo development in mice. Materials and methods: Adult male C57BL/6J mice aged 8–9 weeks were exposed to di(2‐ethylhexyl) phthalate, di‐n‐butyl phthalate, or their mixture (di‐n‐butyl phthalate + di(2‐ethylhexyl) phthalate) at 2.5 mg/kg/day or vehicle for 40 days (equivalent to one spermatogenic cycle) via surgically implanted osmotic pumps. Caudal epididymal spermatozoa were extracted and analyzed for motility using computer‐assisted sperm analyses. Sperm phosphorylation of protein kinase A substrates and tyrosine phosphorylation, markers of early and late capacitation events, respectively, were analyzed by Western blots. In vitro fertilization was used to evaluate the sperm fertilizing capacity. Results: While the study did not reveal any significant differences in sperm motility and fertilization potential, abnormal sperm morphology was observed in all phthalate exposures, particularly in the phthalate mixture group. In addition, the study revealed significant differences in sperm concentration between control and exposed groups. Moreover, protein phosphorylation of protein kinase A substrates was decreased in the di(2‐ethylhexyl) phthalate and mixture exposure groups, while no significant changes in protein tyrosine phosphorylation were observed in any of the groups. Assessment of the reproductive functionality did not reveal significant effects on in vitro fertilization and early embryo development rates but showed wide variability in the phthalate mixture group. Conclusion: Our findings suggest that preconception phthalate exposure affects sperm numbers and phosphorylation of protein kinase A substrates involved in capacitation. Future research is warranted to examine the associations between phthalate exposure and capacitation in human spermatozoa. [ABSTRACT FROM AUTHOR]

Background: Currently, the precise mechanisms that underline male infertility are still unclear. Accumulating data implicate non‐coding RNA cargo of seminal plasma extracellular vesicles due to their association with poor semen quality and higher expression levels relative to vesicle‐free seminal plasma. Method: We assessed sperm‐free seminal plasma extracellular vesicle non‐coding RNA profiles from 91 semen samples collected from male participants of couples seeking infertility treatment. Men were classified into two groups (poor, n = 32; normal, n = 59) based on World Health Organization semen cutoffs. Small RNA sequencing reads were mapped to standard biotype‐specific transcriptomes in the order micro RNA > transfer RNA > piwi‐interacting RNA > ribosomal RNA > ribosomal RNA > circular RNA > long non‐coding RNA using STAR. Differential expression of normalized non‐coding RNA read counts between the two groups was conducted by EdgeR (Fold change ≥1.5 and (false discovery rate [FDR] < 0.05). Result: Small RNA sequencing identified a wide variety of seminal plasma extracellular vesicle non‐coding RNA biotypes including micro RNA, ribosomal RNAs, piwi‐interacting RNAs, transfer RNA, long non‐coding RNAs as well as circular RNAs, and fragments associated with pseudogenes, and nonsense‐mediated decay. The expression levels of 57 seminal plasma extracellular vesicle non‐coding RNAs (micro RNA: 6, piwi‐interacting RNA: 4, ribosomal RNA: 6, circular RNA: 34, and long non‐coding RNA: 7) were altered in men with poor semen quality relative to normal semen parameters, many (60%) of which were circular RNA species. Ontology analysis of differentially expressed micro RNAs and circular RNAs showed enrichment in functional terms related to cellular communication and early development. Conclusion: This is the first study to generate comprehensive seminal plasma extracellular vesicle non‐coding RNA profiles in a clinical setting and to determine the differences between men with normal and abnormal semen parameters. Thus, our study suggests that seminal plasma extracellular vesicle non‐coding RNAs may represent novel biomarkers of male reproductive phenotypes. [ABSTRACT FROM AUTHOR]

Study Question: Is sperm epigenetic aging (SEA) associated with probability of pregnancy among couples in the general population?Summary Answer: We observed a 17% lower cumulative probability at 12 months for couples with male partners in the older compared to the younger SEA categories.What Is Known Already: The strong relation between chronological age and DNA methylation profiles has enabled the estimation of biological age as epigenetic 'clock' metrics in most somatic tissue. Such clocks in male germ cells are less developed and lack clinical relevance in terms of their utility to predict reproductive outcomes.Study Design, Size, Duration: This was a population-based prospective cohort study of couples discontinuing contraception to become pregnant recruited from 16 US counties from 2005 to 2009 and followed for up to 12 months.Participants/materials, Setting, Methods: Sperm DNA methylation from 379 semen samples was assessed via a beadchip array. A state-of-the-art ensemble machine learning algorithm was employed to predict age from the sperm DNA methylation data. SEA was estimated from clocks derived from individual CpGs (SEACpG) and differentially methylated regions (SEADMR). Probability of pregnancy within 1 year was compared by SEA, and discrete-time proportional hazards models were used to evaluate the relations with time-to-pregnancy (TTP) with adjustment for covariates.Main Results and the Role Of Chance: Our SEACpG clock had the highest predictive performance with correlation between chronological and predicted age (r = 0.91). In adjusted discrete Cox models, SEACpG was negatively associated with TTP (fecundability odds ratios (FORs)=0.83; 95% CI: 0.76, 0.90; P = 1.2×10-5), indicating a longer TTP with advanced SEACpG. For subsequent birth outcomes, advanced SEACpG was associated with shorter gestational age (n = 192; -2.13 days; 95% CI: -3.67, -0.59; P = 0.007). Current smokers also displayed advanced SEACpG (P < 0.05). Finally, SEACpG showed a strong performance in an independent IVF cohort (n = 173; r = 0.83). SEADMR performance was comparable to SEACpG but with attenuated effect sizes.Limitations, Reasons For Caution: This prospective cohort study consisted primarily of Caucasian men and women, and thus analysis of large diverse cohorts is necessary to confirm the associations between SEA and couple pregnancy success in other races/ethnicities.Wider Implications Of the Findings: These data suggest that our sperm epigenetic clocks may have utility as a novel biomarker to predict TTP among couples in the general population and underscore the importance of the male partner for reproductive success.Study Funding/competing Interest(s): This work was funded in part by grants the National Institute of Environmental Health Sciences, National Institutes of Health (R01 ES028298; PI: J.R.P. and P30 ES020957); Robert J. Sokol, MD Endowed Chair of Molecular Obstetrics and Gynecology (J.R.P.); and the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland (Contracts N01-HD-3-3355, N01-HD-3-3356 and N01-HD-3-3358). S.L.M. was supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health. The authors declare no competing interests.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Motivation A wide range of computational packages has been developed for regional DNA methylation analyses of Illumina's Infinium array data. Aclust, one of the first unsupervised algorithms, was originally designed to analyze regional methylation of Infinium's 27K and 450K arrays by clustering neighboring methylation sites prior to downstream analyses. However, Aclust relied on outdated packages that rendered it largely non-operational especially with the newer Infinium EPIC and mouse arrays. Results We have created Aclust2.0, a streamlined pipeline that involves five steps for the analyses of human (450K and EPIC) and mouse array data. Aclust2.0 provides a user-friendly pipeline and versatile for regional DNA methylation analyses for molecular epidemiological and mouse studies. Availability and implementation Aclust2.0 is freely available on Github (https://github.com/OluwayioseOA/Alcust2.0.git). [ABSTRACT FROM AUTHOR]

Background: Infertility is a common reproductive disorder, with male factor infertility accounting for approximately half of all cases. Taking a paternal perceptive, recent research has shown that sperm epigenetics, such as changes in DNA methylation, histone modification, chromatin structure, and noncoding RNA expression, can impact reproductive and offspring health. Importantly, environmental conditions during the preconception period has been demonstrated to shape sperm epigenetics. Objectives: To provide an overview on epigenetic modifications that regulate normal gene expression and epigenetic remodeling that occurs during spermatogenesis, and to discuss the epigenetic alterations that may occur to the paternal germline as a consequence of preconception environmental conditions and exposures. Materials and methods: We examined published literature available on databases (PubMed, Google Scholar, ScienceDirect) focusing on adult male preconception environmental exposures and sperm epigenetics in epidemiologic studies and animal models. Results: The preconception period is a sensitive developmental window in which a variety of exposures such as toxicants, nutrition, drugs, stress, and exercise, affects sperm epigenetics. Discussion and Conclusion: Understanding the environmental legacy of the sperm epigenome during spermatogenesis will enhance our understanding of reproductive health and improve reproductive success and offspring well‐being. [ABSTRACT FROM AUTHOR]

We have recently shown that sperm epigenetic age (SEA), a surrogate measure of biological aging in sperm, is associated with couples' time-to-pregnancy (TTP). Advanced SEA was also observed among smokers, suggesting its susceptibility to environmental exposures. Therefore, we assessed the association between urinary phthalate metabolites and SEA in male partners of couples planning to conceive among the general population. The Longitudinal Investigation of Fertility and the Environment (LIFE) Study was a prospective multi-site and general population cohort study of couples who were interested in becoming pregnant. Among male partners (n = 333), eleven urinary phthalate metabolites were measured and SEA was previously developed using Super Learner ensemble algorithm. Multivariable linear regression was used to evaluate associations of SEA with individual metabolites. Bayesian kernel machine regression (BKMR), quantile g-computation (qgcomp) and weighted quantile sum (WQS) models were used for mixture analyses. Covariates included were BMI, cotinine, race and urinary creatinine. In the single metabolite multivariate analyses, nine (82%) phthalate metabolites displayed positive trends with SEA (range: 0.05–0.47 years). Of these metabolites, advanced SEA was significantly associated with interquartile range increases in exposure of three phthalates [MEHHP (β = 0.23, 95% CI: 0.03, 0.43, p = 0.03), MMP (β = 0.24, 95% CI: 0.01, 0.47, p = 0.04), and MiBP (β = 0.47, 95% CI: 0.14, 0.81, p = 0.01)]. Additionally, in BKMR and qgcomp (p = 0.06), but not WQS models, phthalate mixtures showed an overall positive trend with SEA, with MiBP, MMP and MBzP as major drivers of the mixture effects. This is the first study that combined single exposure and mixture models to associate male phthalate exposures with advanced epigenetic aging of sperm in men planning to conceive among the general population. Our findings suggest that phthalate exposure may contribute to the acceleration of biological aging of sperm. • Urinary metabolite concentrations of MEHHP, MiBP, and MMP were positively associated with sperm epigenetic aging (SEA). • There was an overall positive trend between phthalate metabolite mixtures and SEA, with MiBP, MMP and MBzP as major drivers. • Our results suggest that phthalate exposures in adult males contribute to the acceleration of biological aging of sperm. [ABSTRACT FROM AUTHOR]

• Male preconception DEHP exposure in mice modified DNA methylomes in F0 sperm and F1 embryo. • Male preconception DEHP exposure altered F1 embryonic transcriptome at developmental genes. • Spermatogenesis is a sensitive window that may alter F1 development. Preconception environmental conditions have been demonstrated to shape sperm epigenetics and subsequently offspring health and development. Our previous findings in humans showed that urinary anti-androgenic phthalate metabolites in males were associated with altered sperm methylation and blastocyst-stage embryo development. To corroborate this, we examined the effect of preconception exposure to di(2-ethylhexyl) phthalate (DEHP) on genome-wide DNA methylation and gene expression profiles in mice. Eight-week old C57BL/6J male mice were exposed to either a vehicle control, low, or high dose of DEHP (2.5 and 25 mg/kg/weight, respectively) for 67 days (~2 spermatogenic cycles) and were subsequently mated with unexposed females. Reduced representation bisulfite sequencing (RRBS) of epididymal sperm was performed and gastrulation stage embryos were collected for RRBS and transcriptome analyses in both embryonic and extra-embryonic lineages. Male preconception DEHP exposure resulted in 704 differentially methylated regions (DMRs; q-value < 0.05; ≥10% methylation change) in sperm, 1,716 DMRs in embryonic, and 3,181 DMRs in extra-embryonic tissue. Of these, 29 DMRs overlapped between sperm and F1 tissues, half of which showed concordant methylation changes between F0 and F1 generations. F1 transcriptomes at E7.5 were also altered by male preconception DEHP exposure including developmental gene families such as Hox , Gata , and Sox. Additionally, gene ontology analyses of DMRs and differentially expressed genes showed enrichment of multiple developmental processes including embryonic development, pattern specification and morphogenesis. These data indicate that spermatogenesis in adult may represent a sensitive window in which exposure to DEHP alters the sperm methylome as well as DNA methylation and gene expression in the developing embryo. [ABSTRACT FROM AUTHOR]

The well-documented relationship between chronological age and the sperm methylome has allowed for the construction of epigenetic clocks that estimate the biological age of sperm based on DNA methylation, which we previously termed sperm epigenetic age (SEA). Our lab demonstrated that SEA is positively associated with the time taken to achieve pregnancy; however, its relationship with semen parameters is unknown. A total of 379 men from the Longitudinal Investigation of Fertility and Environment (LIFE) study, a non-clinical cohort, and 192 men seeking fertility treatment from the Sperm Environmental Epigenetics and Development Study (SEEDS) were included in the study. Semen analyses were conducted for both cohorts, and SEA was previously generated using a machine learning algorithm and DNA methylation array data. Association analyses were conducted via multivariable linear regression models adjusting for BMI and smoking status. We found that SEA was not associated with standard semen characteristics in SEEDS and LIFE cohorts. However, SEA was significantly associated with higher sperm head length and perimeter, the presence of pyriform and tapered sperm, and lower sperm elongation factor in the LIFE study ( p < 0.05). Based on our results, SEA is mostly associated with defects in sperm head morphological factors that are less commonly evaluated during male infertility assessments. SEA shows promise to be an independent biomarker of sperm quality to assess male fecundity.