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Non-coding RNA (ncRNA) cargo of extracellular vesicles (EVs) in the male reproductive tract play critical roles in semen quality and emerging evidence suggests their susceptibility to environmental factors. Male phthalate exposures have been linked to poor semen quality, sperm DNA methylation profiles and embryo development; however, there is limited evidence on their potential impact on EV ncRNAs profiles. We evaluated the association between urinary phthalate metabolites and small ncRNAs (sncRNAs) of seminal plasma EVs (spEV) among men receiving clinical infertility care. We conducted sncRNA sequencing of EVs in 96 seminal plasma samples collected from the Sperm Environmental Epigenetics and Development Study (SEEDS). Sequencing reads were mapped to human transcriptome databases using STAR. Urinary metabolite concentrations of thirteen phthalates and two DiNCH, a phthalate alternative, were measured via tandem mass spectrometry. Associations with normalized counts were assessed using EdgeR (FDR<0.05) adjusting for urinary dilution via specific gravity, age, BMI, batch, and biotype-specific total counts. Select metabolites, MEOHP, MECPP, ∑DEHP, MCPP, MCNP, MCOP, were negatively (p < 0.05) correlated with miRNA relative abundance. Similarly, nine metabolites including MEOHP, MECPP, MEHP, MCPP, MHBP, MHiNCH, MiBP, MEHHP, MCOP and ∑DEHP were associated (q < 0.05) with normalized counts from 23 unique ncRNA transcripts (7 miRNAs (pre & mature); 6 tRFs; and 10 piRNAs), most (78%) of which displayed increased expression patterns. miRNA and tRFs gene targets were enriched in vesicle-mediated transport and developmental-related ontology terms, such as tyrosine kinase, head development, and cell morphogenesis. Six genes (MAPK1, BMPR1A/2, PTEN, TGFBR2, TP53 and APP) were present in all the ontology terms and predicted to form protein association networks. piRNAs were annotated to pseudogenes of genes important in EV cargo transfer and embryonic development. This is the first study to associate phthalate exposures to altered spEV sncRNA profiles. Future studies are needed to determine their impact on reproductive outcomes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Study questionIs sperm epigenetic aging (SEA) associated with probability of pregnancy among couples in the general population?Summary answerWe 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 alreadyThe 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, durationThis 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, methodsSperm 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 chanceOur 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

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]