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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]

The age-related decline in fat-free mass is accelerated in women after menopause, implying that ovarian hormone deficiency may have catabolic effects on lean tissue. Because fat-free tissue mass is largely determined by its protein content, alterations in ovarian hormones would likely exert regulatory control through effects on protein balance. To address the hypothesis that ovarian hormones regulate protein metabolism, we examined the effect of menopausal status and hormone replacement therapy (HRT) on protein turnover. Whole body protein breakdown, oxidation, and synthesis were measured under postabsorptive conditions using [[sup.13]C]leucine in healthy premenopausal (n = 15, 49 [+ or -] 1 yr) and postmenopausal (n = 18, 53 [+ or -] 1 yr) women. In postmenopausal women, whole body protein turnover and plasma albumin synthesis rates (assessed using [[sup.13]C]leucine and [[sup.2]H]phenylalanine) were also measured following 2 mo of treatment with oral HRT (0.625 mg conjugated estrogens + 2.5 mg medroxyprogesterone acetate, n = 9) or placebo (n = 9). No differences in whole body protein breakdown, oxidation, or synthesis were found between premenopausal and postmenopausal women. Protein metabolism remained similar between groups after statistical adjustment for differences in adiposity and when subgroups of women matched for percent body fat were compared. In postmenopausal women, no effect of HRT was found on whole body protein breakdown, synthesis, or oxidation. In contrast, our results support a stimulatory effect of HRT on albumin fractional synthesis rate, although this did not translate into alterations in circulating albumin concentrations. In conclusion, our results suggest no detrimental effect of ovarian hormone deficiency coincident with the postmenopausal state, and no salutary effect of hormone repletion with HRT, on rates of whole body protein turnover, although oral HRT regimens may increase the synthesis rates of albumin. menopause; estrogen; progesterone; fat-free mass; sarcopenia doi:10.1152/ajpendo.00050.2006

The age-related decline in fat-free mass is accelerated in women after menopause. The role of ovarian hormone deficiency in the regulation of fat-free mass, however, has not been clearly defined. To address this question, we examined the effect of ovarian hormone suppression on whole body protein metabolism. Whole body protein breakdown, oxidation, and synthesis were measured using [[sup.13]C]leucine in young, healthy women with regular menstrual patterns before and after 2 mo of treatment with gonadotropin-releasing hormone agonist (GnRHa; n = 6) or placebo (n = 7). Protein metabolism was measured under postabsorptive and euglycemic-hyperinsulinemic-hyperaminoacidemic conditions. Ovarian suppression did not alter whole body or regional fat-free mass or adiposity. In the postabsorptive state, GnRHa administration was associated with reductions in protein breakdown and synthesis (P < 0.05), whereas no change in protein oxidation was noted. Under euglycemic-hyperinsulinemic-hyperaminoacidemic conditions, a similar reduction (P < 0.05) in protein synthesis and breakdown was noted, whereas, protein oxidation increased (P < 0.05) in the placebo group. Testosterone, steroid hormone precursors, insulin-like growth factor I, and their respective binding proteins were not altered by GnRHa administration, and changes in these hormones over time were not associated with GnRHa-induced alterations in protein metabolism, suggesting that changes in protein turnover are not due to an effect of ovarian suppression on other endocrine systems. Our findings provide evidence that endogenous ovarian hormones participate in the regulation of protein turnover in women. estrogen; menopause; fat-free mass; sarcopenia; progesterone doi: 10.1152/ajpendo.00600.2005

Study Question: Are sperm mitochondrial DNA copy number (mtDNAcn) and deletion rate (mtDNAdel) associated with odds of fertilization and high embryo quality at Days 3 and 5?Summary Answer: Higher sperm mtDNAcn and mtDNAdel were associated with lower odds of high quality Day 3 embryos and transfer quality Day 5 embryos, both of which were primarily driven by lowered odds of fertilization.What Is Known Already: Sperm mtDNAcn and mtDNAdel have been previously associated with poor semen parameters and clinical male infertility. One prior study has shown that mtDNAdel is associated with lower fertilization rates. However, it is unknown whether these characteristics are linked with ART outcomes.Study Design, Size, Duration: This prospective observational study included 119 sperm samples collected from men undergoing ART in Western Massachusetts. ART outcomes were observed through to Day 5 post-insemination.Participants/materials, Settings, Methods: As part of the Sperm Environmental Epigenetics and Development Study (SEEDS), 119 sperm samples were collected from men undergoing ART in Western Massachusetts. Sperm mtDNAcn and mtDNAdel were measured via triplex probe-based qPCR. Fertilization, Day 3 embryo quality and Day 5 embryo quality measures were fitted with mtDNAcn and mtDNAdel using generalized estimating equations.Main Results and the Role Of Chance: After adjusting for male age and measurement batches, higher sperm mtDNAcn and mtDNAdel were associated with lower odds of fertilization (P = 0.01 and P < 0.01), high quality Day 3 embryos (P = 0.02 for both) and transfer quality Day 5 embryos (P = 0.01 and P = 0.09). However, the associations of mtDNAcn and mtDNAdel with Day 3 high quality status and Day 5 transfer quality status were attenuated in models restricted to fertilized oocytes. Sperm mtDNAcn and mtDNAdel remained statistically significant in models adjusted for both male age and semen parameters, although models including both mtDNA markers generally favoured mtDNAdel.Limitations, Reasons For Caution: Our sample only included oocytes and embryos from 119 couples and thus large diverse cohorts are necessary to confirm the association of sperm mtDNA biomarkers with embryo development.Wider Implications Of the Findings: To our knowledge, our study is the first to assess the associations of sperm mtDNAcn and mtDNAdel with fertilization and embryo quality. The biological mechanism(s) underlying these associations are unknown. Multivariable models suggest that sperm mtDNAcn and mtDNAdel provide discrimination independent of age and semen parameters; therefore, future investigation of the utility of sperm mtDNA as a biomarker for ART outcomes is warranted.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.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Determinants of insulin-stimulated glucose disposal in middle-aged, premenopausal women. Am J Physiol Endocrinol Metab 281: E113-E121, 2001.--Controversy exists regarding the relative importance of adiposity, physical fitness, and physical activity in the regulation of insulin-stimulated glucose disposal. To address this issue, we measured insulin-stimulated glucose disposal [mg [multiplied by] kg fat-free mass [(FFM).sup.-1] [multiplied by] [min.sup.-1]; oxidative and nonoxidative components] in 45 nondiabetic, nonobese, premenopausal women (mean [+ or -] SD; 47 [+ or -] 3 yr) by use of hyperinsulinemic euglycemic clamp (40 mU [multiplied by] [m.sup.-2]. [min.sup.-1]) and [6,6-[sup.2][H.sub.2]]glucose dilution techniques. We also measured body composition, abdominal fat distribution, thigh muscle fat content, maximal oxygen consumption ([VO.sub.2 max]), and physical activity energy expenditure ([sup.2][H.sub.2][sup.18]O kinetics) as possible correlates of glucose disposal. [VO.sub.2 max] was the strongest correlate of glucose disposal (r = 0.63, P [is less than] 0.01), whereas whole body and abdominal adiposity showed modest associations (range of r values from -0.32 to -0.46, P [is less than] 0.05 to P [is less than] 0.01). A similar pattern of correlations was observed for nonoxidative glucose disposal. None of the variables measured correlated with oxidative glucose disposal. The relationship of [VO.sub.2 max] to glucose disposal persisted after statistical control for FFM, percent body fat, and intra-abdominal fat (r = 0.40, P [is less than] 0.01). In contrast, correlations of total and regional adiposity measures to insulin sensitivity were no longer significant after statistical adjustment for [VO.sub.2 max]. [VO.sub.2 max] was the only variable to enter stepwise regression models as a significant predictor of total and nonoxidative glucose disposal. Our results highlight the importance of [VO.sub.2 max] as a determinant of glucose disposal and suggest that it may be a stronger determinant of variation in glucose disposal than total and regional adiposity in nonobese, nondiabetic, premenopausal women. aerobic fitness; body composition; body fat distribution; euglycemic clamp

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. [ABSTRACT FROM AUTHOR]

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 Center's 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 sexspecific 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. [ABSTRACT FROM AUTHOR]