Tayyab Rahil

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.

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.

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.

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 embryos ability to produce beta-HCG, but that vitrification does not impair developmental potential.

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.