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 (ß = 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.

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.

Implantation of fresh and thawed-warmed embryos in single embryo transfer cycles: interpreting the initial beta-HCG.

Little is known about the effects of human embryo cryopreservation on developmental potential. Initial beta-HCG, indicating embryo implantation, was measured in 322 single embryo transfer cycles (246 fresh and 76 thawed-warmed). Median initial beta-HCG was higher for fresh compared with thawed-warmed transfers (126 versus 100 mIU/ml; P = 0.04). Blastocyst slow cooling resulted in a lower initial beta-HCG compared with vitrification (P = 0.01). Live birth rates were lower for blastocyst slow cooling (25%) compared with vitrification (71%) and fresh transfer (70%). We conclude that cryopreservation may impair an embryo's ability to produce beta-HCG, but that vitrification does not impair developmental potential.

Array comparative genomic hybridization screening in IVF significantly reduces number of embryos available for cryopreservation.

OBJECTIVE: During IVF, non-transferred embryos are usually selected for cryopreservation on the basis of morphological criteria. This investigation evaluated an application for array comparative genomic hybridization (aCGH) in assessment of surplus embryos prior to cryopreservation. METHODS: First-time IVF patients undergoing elective single embryo transfer and having at least one extra non-transferred embryo suitable for cryopreservation were offered enrollment in the study. Patients were randomized into two groups: Patients in group A (n=55) had embryos assessed first by morphology and then by aCGH, performed on cells obtained from trophectoderm biopsy on post-fertilization day 5. Only euploid embryos were designated for cryopreservation. Patients in group B (n=48) had embryos assessed by morphology alone, with only good morphology embryos considered suitable for cryopreservation. RESULTS: Among biopsied embryos in group A (n=425), euploidy was confirmed in 226 (53.1%). After fresh single embryo transfer, 64 (28.3%) surplus euploid embryos were cryopreserved for 51 patients (92.7%). In group B, 389 good morphology blastocysts were identified and a single top quality blastocyst was selected for fresh transfer. All group B patients (48/48) had at least one blastocyst remaining for cryopreservation. A total of 157 (40.4%) blastocysts were frozen in this group, a significantly larger proportion than was cryopreserved in group A (p=0.017, by chi-squared analysis). CONCLUSION: While aCGH and subsequent frozen embryo transfer are currently used to screen embryos, this is the first investigation to quantify the impact of aCGH specifically on embryo cryopreservation. Incorporation of aCGH screening significantly reduced the total number of cryopreserved blastocysts compared to when suitability for freezing was determined by morphology only. IVF patients should be counseled that the benefits of aCGH screening will likely come at the cost of sharply limiting the number of surplus embryos available for cryopreservation.