Intraindividual variability of semen quality, proteome, and sncRNA profiles in a healthy cohort of young adults.

BACKGROUND: Within-subject variability of semen parameters and molecular components of ejaculates in young men remains poorly understood. OBJECTIVES: To investigate intraindividual variability (IIV) of semen parameters and molecular markers in repeated ejaculates from young men. MATERIALS AND METHODS: Semen parameters were assessed in samples collected 6-8 days apart from 164 18-19-year old participants of the Russian Children's Study, a prospective cohort. Subsets of paired samples were used for label-free quantitation and targeted mass-spectrometry of proteins in seminal plasma (SP) and seminal extracellular vesicles (EVs), and for small non-coding RNA (sncRNA) profiling in EVs and spermatozoa using RNA-seq. The mean difference between two ejaculates, within-subject variation, intraclass correlation, and concordance correlation were used to assess IIV for all parameters. Low variability with high reproducibility and high reliability was considered if CVw < 15% and ICC > 0.90, respectively. RESULTS: Analytical variability was low for all investigated parameters in technical replicates. IIV was assessed for basic semen parameters and proteins in SPs and EVs: 319 and 777 proteins, respectively, using untargeted analysis; 9 and 10 proteins using targeted quantification. We also described the IIV for sncRNA, including microRNA, piwi-interacting RNA, tRNA, and tRNA-derived small RNA (tsRNA) in EVs (409 sncRNA and 78 tsRNA) and in spermatozoa (265 sncRNA and 15 tsRNA). We identified 22 and 27 non-overlapping proteins in SP and EVs, respectively, and 46 and 9 sncRNA, including 5 and 0 tsRNA in seminal EVs and spermatozoa, respectively, with low variability. The fatty acid synthase (FAS) had the lowest IIV in both media in targeted protein quantification. DISCUSSION: We identified a number of proteins and sncRNA with low variability among 111 proteins, 176 sncRNA, and 12 tsRNA which were previously suggested as biomarkers of male fertility and reproductive outcomes: lactotransferrin, cysteine-rich secretory protein 3, alpha-1-antichymotrypsin, epididymal sperm-binding protein 1, glutathione S-transferase Mu 3, alpha-1-acid glycoprotein 2, serum amyloid P-component, aminopeptidase N, neprilysin, FAS, and miR-10b-3p, miR-122-5p, miR-205-5p, miR-222-3p, miR-34c-5p, miR-509-3-5p, miR-888-5p, miR-892a, miR-363-3p, miR-941, miR-146a-5p, miR-744-5p. CONCLUSION: These molecules have low IIV and may be promising candidate biomarkers of male fertility and reproductive health.

Epigenetic age acceleration in follicular fluid and markers of ovarian response among women undergoing IVF.

STUDY QUESTION: Are markers of epigenetic age acceleration in follicular fluid associated with outcomes of ovarian stimulation? SUMMARY ANSWER: Increased epigenetic age acceleration of follicular fluid using the Horvath clock, but not other epigenetic clocks (GrimAge and Granulosa Cell), was associated with lower peak estradiol levels and decreased number of total and mature oocytes. WHAT IS KNOWN ALREADY: In granulosa cells, there are inconsistent findings between epigenetic age acceleration and ovarian response outcomes. STUDY DESIGN, SIZE, DURATION: Our study included 61 women undergoing IVF at an academic fertility clinic in the New England area who were part of the Environment and Reproductive Health Study (2006-2016). PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants provided a follicular fluid sample during oocyte retrieval. DNA methylation of follicular fluid was assessed using a genome-wide methylation screening tool. Three established epigenetic clocks (Horvath, GrimAge, and Granulosa Cell) were used to predict DNA-methylation-based epigenetic age. To calculate the age acceleration, we regressed epigenetic age on chronological age and extracted the residuals. The association between epigenetic age acceleration and ovarian response outcomes (peak estradiol levels, follicle stimulation hormone, number of total, and mature oocytes) was assessed using linear and Poisson regression adjusted for chronological age, three surrogate variables (to account for cellular heterogeneity), race, smoking status, initial infertility diagnosis, and stimulation protocol. MAIN RESULTS AND ROLE OF CHANCE: Compared to the median chronological age of our participants (34 years), the Horvath clock predicted, on an average, a younger epigenetic age (median: 24.2 years) while the GrimAge (median: 38.6 years) and Granulosa Cell (median: 39.0 years) clocks predicted, on an average, an older epigenetic age. Age acceleration based on the Horvath clock was associated with lower peak estradiol levels (-819.4 unit decrease in peak estradiol levels per standard deviation increase; 95% CI: -1265.7, -373.1) and fewer total (% change in total oocytes retrieved per standard deviation increase: -21.8%; 95% CI: -37.1%, -2.8%) and mature oocytes retrieved (% change in mature oocytes retrieved per standard deviation increase: -23.8%; 95% CI: -39.9%, -3.4%). The age acceleration based on the two other epigenetic clocks was not associated with markers of ovarian response. LIMITATIONS, REASONS FOR CAUTION: Our sample size was small and we did not specifically isolate granulosa cells from follicular fluid samples so our samples could have included mixed cell types. WIDER IMPLICATIONS OF THE FINDINGS: Our results highlight that certain epigenetic clocks may be predictive of ovarian stimulation outcomes when applied to follicular fluid; however, the inconsistent findings for specific clocks across studies indicate a need for further research to better understand the clinical utility of epigenetic clocks to improve IVF treatment. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by grants ES009718, ES022955, ES000002, and ES026648 from the National Institute of Environmental Health Sciences (NIEHS) and a pilot grant from the NIEHS-funded HERCULES Center at Emory University (P30 ES019776). RBH was supported by the Emory University NIH Training Grant (T32-ES012870). TRIAL REGISTRATION NUMBER: N/A.

Phthalates and sex steroid hormones across the perimenopausal period: A longitudinal analysis of the Midlife Women's Health Study.

BACKGROUND: The menopausal transition involves significant sex hormone changes. Environmental chemicals, such as urinary phthalate metabolites, are associated with sex hormone levels in cross-sectional studies. Few studies have assessed longitudinal associations between urinary phthalate metabolite concentrations and sex hormone levels during menopausal transition. METHODS: Pre- and perimenopausal women from the Midlife Women's Health Study (MWHS) (n = 751) contributed data at up to 4 annual study visits. We quantified 9 individual urinary phthalate metabolites and 5 summary measures (e.g., phthalates in plastics (∑Plastic)), using pooled annual urine samples. We measured serum estradiol, testosterone, and progesterone collected at each study visit, unrelated to menstrual cycling. Linear mixed-effects models and hierarchical Bayesian kernel machine regression analyses evaluated adjusted associations between individual and phthalate mixtures with sex steroid hormones longitudinally. RESULTS: We observed associations between increased concentrations of certain phthalate metabolites and lower testosterone and higher sub-ovulatory progesterone levels, e.g., doubling of monoethyl phthalate (MEP), monobenzyl phthalate (MBzP), di-2-ethylhexyl phthalate (∑DEHP) metabolites, ∑Plastic, and ∑Phthalates concentrations were associated with lower testosterone (e.g., for ∑DEHP: -4.51%; 95% CI: -6.72%, -2.26%). For each doubling of MEP, certain DEHP metabolites, and summary measures, we observed higher mean sub-ovulatory progesterone (e.g., ∑AA (metabolites with anti-androgenic activity): 6.88%; 95% CI: 1.94%, 12.1%). Higher levels of the overall time-varying phthalate mixture were associated with lower estradiol and higher progesterone levels, especially for 2nd year exposures. CONCLUSIONS: Phthalates were longitudinally associated with sex hormone levels during the menopausal transition. Future research should assess such associations and potential health impacts during this understudied period.

The associations between pre-conception urinary phthalate concentrations, the serum metabolome, and live birth among women undergoing assisted reproduction.

BACKGROUND: Phthalates are ubiquitous endocrine disruptors. Past studies have shown an association between higher preconception urinary concentrations of phthalate metabolites and lower fertility in women; however, the biological mechanisms remain unclear. Our exploratory study aimed to understand the metabolites and pathways associated with maternal preconception phthalate exposure and examine if any may underline the association between phthalate exposure and live birth using untargeted metabolomics. METHODS: Participants (n = 183) were part of the Environment and Reproductive Health (EARTH) study, a prospective cohort that followed women undergoing in vitro fertilization (IVF) at the Massachusetts General Hospital Fertility Center (2005-2016). On the same day, women provided a serum sample during controlled ovarian stimulation, which was analyzed for metabolomics using liquid chromatography coupled with high-resolution mass spectrometry and two chromatography columns, and a urine sample, which was analyzed for 11 phthalate metabolites using targeted approaches. We used multivariable generalized linear models to identified metabolic features associated with urinary phthalate metabolite concentrations and live birth, followed by enriched pathway analysis. We then used a meet-in-the-middle approach to identify overlapping pathways and features. RESULTS: Metabolic pathway enrichment analysis revealed 43 pathways in the C18 negative and 32 pathways in the HILIC positive columns that were significantly associated (p < 0.05) with at least one of the 11 urinary phthalate metabolites or molar sum of di-2-ethylhexyl phthalate metabolites. Lipid, amino acid, and carbohydrate metabolism were the most common pathways associated with phthalate exposure. Five pathways, tryptophan metabolism, tyrosine metabolism, biopterin metabolism, carnitine shuttle, and vitamin B6 metabolism, were also identified as being associated with at least one phthalate metabolite and live birth following IVF. CONCLUSION: Our study provides further insight into the metabolites and metabolomics pathways, including amino acid, lipid, and vitamin metabolism that may underlie the observed associations between phthalate exposures and lower fertility in women.

Irregular Cycles, Ovulatory Disorders, and Cardiometabolic Conditions in a US-Based Digital Cohort.

Importance: Polycystic ovary syndrome (PCOS), characterized by irregular menstrual cycles and hyperandrogenism, is a common ovulatory disorder. Having an irregular cycle is a potential marker for cardiometabolic conditions, but data are limited on whether the associations differ by PCOS status or potential interventions. Objective: To evaluate the association of PCOS, time to regularity since menarche (adolescence), and irregular cycles (adulthood) with cardiometabolic conditions. Design, Setting, and Participants: This cross-sectional study used a large, US-based digital cohort of users of the Apple Research application on their iPhone. Eligibility criteria were having ever menstruated, living in the US, being at age of consent of at least 18 years (or 19 years in Alabama and Nebraska or 21 years in Puerto Rico), and being able to communicate in English. Participants were enrolled between November 14, 2019, and December 13, 2022, and completed relevant surveys. Exposures: Self-reported PCOS diagnosis, prolonged time to regularity (not spontaneously establishing regularity within 5 years of menarche), and irregular cycles. Main Outcomes and Measures: The primary outcome was self-reported cardiometabolic conditions, including obesity, prediabetes, type 1 and 2 diabetes, high cholesterol, hypertension, metabolic syndrome, arrhythmia, congestive heart failure, coronary artery disease, heart attack, heart valve disease, stroke, transient ischemic attack (TIA), deep vein thrombosis, and pulmonary embolism measured using descriptive statistics and logistic regression to estimate prevalence odds ratios (PORs) and 95% CIs. Effect modification by lifestyle factors was also estimated. Results: The study sample (N = 60 789) had a mean (SD) age of 34.5 (11.1) years, with 12.3% having PCOS and 26.3% having prolonged time to regularity. Among a subset of 25 399 participants who completed the hormonal symptoms survey, 25.6% reported irregular cycles. In covariate-adjusted logistic regression models, PCOS was associated with a higher prevalence of all metabolic and several cardiovascular conditions, eg, arrhythmia (POR, 1.37; 95% CI, 1.20-1.55), coronary artery disease (POR, 2.92; 95% CI, 1.95-4.29), heart attack (POR, 1.79; 95% CI, 1.23-2.54), and stroke (POR, 1.66; 95% CI, 1.21-2.24). Among participants without PCOS, prolonged time to regularity was associated with type 2 diabetes (POR, 1.24; 95% CI, 1.05-1.46), hypertension (POR, 1.09; 95% CI, 1.01-1.19), arrhythmia (POR, 1.20; 95% CI, 1.06-1.35), and TIA (POR, 1.33; 95% CI, 1.01-1.73), and having irregular cycles was associated with type 2 diabetes (POR, 1.36; 95% CI, 1.08-1.69), high cholesterol (POR, 1.17; 95% CI, 1.05-1.30), arrhythmia (POR, 1.21; 95% CI, 1.02-1.43), and TIA (POR, 1.56; 95% CI, 1.06-2.26). Some of these associations were modified by high vs low body mass index or low vs high physical activity. Conclusions and Relevance: These findings suggest that PCOS and irregular cycles may be independent markers for cardiometabolic conditions. Early screening and intervention among individuals with irregular menstrual cycles may be beneficial.

Endocrine-disrupting chemical concentrations in follicular fluid and follicular reproductive hormone levels.

PURPOSE: To determine correlations between chemicals in follicular fluid (FF) and follicular reproductive hormone levels. METHODS: The analysis was part of a larger cohort study to determine associations between exposure to EDCs and in vitro fertilization (IVF) outcomes. FF was aspirated from a single leading follicle per participant. Demographics and data on exposure to EDCs were self-reported by the participants using a questionnaire. The concentrations of estradiol (E2), progesterone (PG), anti-Mullerian hormone (AMH), and inhibin B, as well as that of 12 phthalate metabolites and 12 phenolic chemicals were measured in each FF sample. Multivariate linear regression model was used to identify the drivers of hormone levels based on participant's age, BMI, smoking status, and chemical exposure for the monitored chemicals detected in more than 50% of the samples. Benjamini-Hochberg false discovery rate (FDR) correction was applied on the resulting p values (q value). RESULTS: FF samples were obtained from 72 women (mean age 30.9 years). Most of the phthalates and phenolic substances monitored (21/24, 88%) were identified in FF. Ten compounds (7 phthalate metabolites, 3 phenols) were found in more than 50% of samples. In addition, there were positive associations between E2 levels and mono-n-butyl phthalate (MnBP) (beta = 0.01) and mono-isobutyl phthalate (MiBP) (beta = 0.03) levels (q value < 0.05). CONCLUSION: Higher concentrations of several phthalate metabolites, present among others in personal care products, were associated with increased E2 levels in FF. The results emphasize the need to further investigate the mechanisms of action of such EDCs on hormonal cyclicity and fertility in women.

Former smoking associated with epigenetic modifications in human granulosa cells among women undergoing assisted reproduction.

Smoking exposure during adulthood can disrupt oocyte development in women, contributing to infertility and possibly adverse birth outcomes. Some of these effects may be reflected in epigenome profiles in granulosa cells (GCs) in human follicular fluid. We compared the epigenetic modifications throughout the genome in GCs from women who were former (N = 15) versus never smokers (N = 44) undergoing assisted reproductive technologies (ART). This study included 59 women undergoing ART. Smoking history including time since quitting was determined by questionnaire. GCs were collected during oocyte retrieval and DNA methylation (DNAm) levels were profiled using the Infinium MethylationEPIC BeadChip. We performed an epigenome-wide association study with robust linear models, regressing DNAm level at individual loci on smoking status, adjusting for age, ovarian stimulation protocol, and three surrogate variables. We performed differentially methylated regions (DMRs) analysis and over-representation analysis of the identified CpGs and corresponding gene set. 81 CpGs were differentially methylated among former smokers compared to never smokers (FDR < 0.05). We identified 2 significant DMRs (KCNQ1 and RHBDD2). The former smoking-associated genes were enriched in oxytocin signaling, adrenergic signaling in cardiomyocytes, platelet activation, axon guidance, and chemokine signaling pathway. These epigenetic variations have been associated with inflammatory responses, reproductive outcomes, cancer development, neurodevelopmental disorder, and cardiometabolic health. Secondarily, we examined the relationships between time since quitting and DNAm at significant CpGs. We observed three CpGs in negative associations with the length of quitting smoking (p < 0.05), which were cg04254052 (KCNIP1), cg22875371 (OGDHL), and cg27289628 (LOC148145), while one in positive association, which was cg13487862 (PLXNB1). As a pilot study, we demonstrated epigenetic modifications associated with former smoking in GCs. The study is informative to potential biological pathways underlying the documented association between smoking and female infertility and biomarker discovery for smoking-associated reproductive outcomes.

Cotinine concentrations in maternal serum and amniotic fluid during pregnancy and risk of testicular germ cell cancer in the offspring: A prospective nested case-control study.

Maternal smoking in pregnancy may increase the risk of testicular germ cell cancer (TGCC) in offspring, but current evidence remains inconclusive. We performed a nested case-control study using cotinine measurements in maternal serum and amniotic fluid as a biomarker for tobacco exposure during pregnancy. A total of 654 males with maternal serum (n = 359, ncases/controls = 71/288) and/or amniotic fluid (n = 295, ncases/controls = 66/229) samples were included. Data on TGCC diagnoses and relevant covariates were derived from nationwide Danish health registries. Cotinine was quantified by liquid chromatography tandem mass spectrometry. An adapted cox regression model estimated the risk of TGCC considering active and inactive tobacco use defined according to cotinine concentrations of <, ≥15 ng/ml. Overall, the concentrations of cotinine were comparable in maternal serum and amniotic fluid (medianserum/amniotic fluid : 2.1/2.6 ng/ml). A strong statistically significant correlation was detected in 14 paired samples (Spearman rho: 0.85). Based on maternal serum cotinine concentrations, exposure to active tobacco use was not associated with risk of TGCC in offspring (HR 0.88, 95% CI 0.51; 1.52). Similarly, based on amniotic fluid cotinine concentrations, exposure to active tobacco use was not associated with risk of TGCC (HR 1.11, 95% CI 0.64; 1.95). However, different risks were observed for seminomas and nonseminomas in both matrices, but none were statistically significant. Our findings did not provide convincing evidence supporting that exposure to tobacco during pregnancy is associated with TGCC.

Serum and follicular fluid metabolome and markers of ovarian stimulation.

STUDY QUESTION: What metabolic pathways and metabolites in the serum and follicular fluid are associated with peak estradiol levels and the number of mature oocytes? SUMMARY ANSWER: In the serum metabolome, mostly fatty acid and amino acid pathways were associated with estradiol levels and mature oocytes while in the follicular fluid metabolome, mostly lipid, vitamin, and hormone pathways were associated with peak estradiol levels and mature oocytes. WHAT IS KNOWN ALREADY: Metabolomics has identified several metabolic pathways and metabolites associated with infertility but limited data are available for ovarian stimulation outcomes. STUDY DESIGN, SIZE, DURATION: A prospective cohort study of women undergoing IVF from 2009 to 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 125 women undergoing a fresh IVF cycle at a fertility clinic in the Northeast United States who provided a serum and follicular fluid sample. Untargeted metabolomics profiling was conducted using liquid chromatography with high-resolution mass spectrometry in two chromatography columns (C18 and hydrophilic interaction chromatography (HILIC)). The main ovarian stimulation outcomes were peak serum estradiol levels and number of mature oocytes. We utilized adjusted generalized linear regression models to identify significant metabolic features. Models were adjusted for age,BMI, initial infertility diagnosis, and ovarian stimulation protocol. We then conducted pathway analysis using mummichog and metabolite annotation using level-1 evidence. MAIN RESULTS AND ROLE OF CHANCE: In the serum metabolome, 480 and 850 features were associated with peak estradiol levels in the C18 and HILIC columns, respectively. Additionally, 437 and 538 features were associated with mature oocytes in the C18 and HILIC columns, respectively. In the follicular fluid metabolome, 752 and 929 features were associated with peak estradiol levels in the C18 and HILIC columns, respectively, Additionally, 993 and 986 features were associated with mature oocytes in the C18 and HILIC columns, respectively. The most common pathways associated with peak estradiol included fatty acids (serum and follicular fluid), hormone (follicular fluid), and lipid pathways (follicular fluid). The most common pathways associated with the number of mature oocytes retrieved included amino acids (serum), fatty acids (serum and follicular fluid), hormone (follicular fluid), and vitamin pathways(follicular fluid). The vitamin D3 pathway had the strongest association with both ovarian stimulation outcomes in the follicularfluid. Four and nine metabolites were identified using level-1 evidence (validated identification) in the serum and follicular fluid metabolomes, respectively. LIMITATIONS, REASONS FOR CAUTION: Our sample was majority White and highly educated and may not be generalizable to thewider population. Additionally, residual confounding is possible and the flushing medium used in the follicular fluid could have diluted our results. WIDER IMPLICATIONS OF THE FINDINGS: The pathways and metabolites identified by our study provide novel insights into the biologicalmechanisms in the serum and follicular fluid that may underlie follicular and oocyte development, which could potentially be used to improve ovarian stimulation outcomes. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the following grants from the National Institute of Environmental Health Sciences (P30-ES019776, R01-ES009718, R01-ES022955, P30-ES000002, R00-ES026648, and T32-ES012870), and National Institute of Diabetes and Digestive and Kidney Diseases (P30DK046200). The authors have no competing interests to disclose. TRIAL REGISTRATION NUMBER: N/A.

Phthalates, adipates, BPA, and pesticides in school meals.

BACKGROUND: Exposure to phthalates, adipates, bisphenol-A (BPA), and pesticides may have important health consequences for children, but little is known regarding their presence in school meals, a major food source for children. The aims of this study were to determine the presence of phthalates, adipates, BPA, and pesticides in school meals. METHODS: Using a cross-sectional design, n = 50 school meal components were collected from four school districts in New England (n = 8 elementary/K-8 schools) differing preparation methods (on-site scratch cooking and pre-packaged heat and serve meals with plastic films) between 2019 and 2021. Meal components were tested for the presence of phthalates, adipates, BPA, and pesticides by an independent laboratory. RESULTS: One adipate, DEHA, was detected in 42% of samples (range 0.08 mg/kg - 50.39 mg/kg). Among the prepackaged foods, DEHA was detected in 86% of samples, which also contained the highest average concentrations among all the samples tested. The phthalate Diisononyl phthalate (DINP) was detected in only one sample, and BPA was not detected in any of the samples tested. Pesticides were detected in 64% of produce and 27% of entrées, but most had levels that were lower than the average levels detected by the USDA's Pesticide Data Program and only one sample had levels that exceeded those detected by the USDA (Cyfluthrin levels in one sample of apples were 0.038 mg/kg compared with an USDA average range of 0.004-0.032 mg/kg). CONCLUSIONS: Overall pesticides and phthalate levels in school meals are low and BPA was not detected. However, this study suggests that manufacturers may be changing to less studied plasticizers such as DEHA. More studies should examine the impact of DEHA on human health, particularly among children. As schools transition back from the COVID-19 pandemic, foods packaged in plastic should be minimized where possible. Overall, school meals remain one of the healthiest options for children and policies that support on site cooking can further strengthen the quality of school meals.

Perceived stress and markers of ovarian reserve among subfertile women.

RESEARCH QUESTION: Is self-reported psychological stress associated with markers of ovarian reserve among subfertile women? DESIGN: Observational study of women (n = 520) seeking fertility care at the Massachusetts General Hospital who enrolled in the Environment and Reproductive Health study between 2005 and 2019. Women completed the short version of the validated PSS4, which assesses psychological stress. Ovarian reserve markers included AFC and circulating serum levels of day-3 FSH, with AMH assessed in a subset of participants (n = 185). RESULTS: Higher total PSS4 scores were negatively associated with AFC and serum AMH levels. Analyses adjusted for age, BMI, race, smoking, education, physical activity and type of infertility diagnosis. Women in the second and third tertiles of stress had lower AFC (13.3, 95% CI 12.7 to 13.8; and 13.5, 95% CI 13.0 to 14.1) compared with women in the lowest tertile of psychological stress score (14.3, 95% CI 13.8 to 14.9, both P < 0.05). Women in the second and third tertiles of total PSS4 scores also had lower mean serum AMH compared with women in the lowest tertile (2.99, 95% CI 2.24 to 3.74), and (2.99 95% CI 2.22 to 3.76) versus (3.94 95% CI 3.23 to 4.64). These associations varied by several socioeconomic factors, and were observed among women who were younger, belonging to minority races, with a college degree or with annual household income less than $100,000. CONCLUSIONS: Higher perceived stress was negatively associated with AFC and serum AMH levels. These associations varied by several socioeconomic factors.

Maternal Exposure to Cigarette Smoke during Pregnancy and Testicular Cancer in Offspring: A Systematic Review and Meta-Analysis.

BACKGROUND: Maternal exposure to cigarette smoke in pregnancy may play a role in the development of testicular cancer in offspring. An updated and comprehensive systematic review of the available evidence is needed. OBJECTIVE: To identify and evaluate current evidence on maternal exposure to cigarette smoke during pregnancy and testicular cancer in offspring. METHODS: A systematic search of English peer-reviewed original literature in PubMed through a block search approach. Publications were considered if assessing maternal exposure to cigarette smoke and the risk of testicular cancer in offspring. RESULTS: Among the 636 identified records, 14 publications were eligible for review and 10 for meta-analysis. Quality assessment of the publications was conducted. Most included publications were case-control studies (n = 11, 79%), while the remaining were ecological studies (n = 3, 21%). Completeness of reporting was high, but more than half were considered subject to potential bias. The trend synthesis showed that half (n = 7) of the included publications demonstrated a higher risk of testicular cancer in the sons of mothers exposed to cigarette smoke during pregnancy. The meta-analysis generated an overall summary risk estimate of 1.00 (95% CI: 0.88; 1.15) (n = 10 publications), with a lower risk for seminoma (0.79, 95% CI: 0.59; 1.04) and nonseminoma (0.96, 95% CI: 0.74; 1.26) (n = 4 publications). CONCLUSIONS: This systematic review did not provide evidence of an association between maternal exposure to cigarette smoke and risk of testicular cancer in offspring. An overall positive trend was suggested, but it had low statistical precision. The methodological limitations across publications encourage further research based on valid exposure data.

Occupational factors and markers of testicular function among men attending a fertility center.

STUDY QUESTION: Are occupational factors associated with markers of testicular function among men attending a fertility center? SUMMARY ANSWER: Men working non-daytime/rotating shifts and those with physically demanding jobs have higher sperm concentration and total sperm count as well as higher estradiol and total testosterone concentrations. WHAT IS KNOWN ALREADY: Semen quality has declined during recent decades and has been negatively correlated with higher risks of common chronic diseases and mortality, highlighting its public health importance beyond fertility and reproduction. While most of the previous epidemiology literature on male fertility has focused on environmental exposures, dietary factors, and other related variables, little attention has been paid to occupational factors. STUDY DESIGN, SIZE, DURATION: This observational study included 377 men who were male partners in couples seeking infertility treatment at a fertility center, who enrolled in the Environment and Reproductive Health (EARTH) study between 2005 and 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS: Self-reported information on lifting/moving heavy objects, typical shift, and physical level of exertion at work was collected from a take-home questionnaire. Semen samples were analyzed following World Health Organization guidelines. Enzyme immunoassays were used to assess reproductive hormone concentrations. Linear regression models were used to evaluate the association between occupational factors and measures of testicular function, while adjusting for covariates such as age, BMI, education, race, smoking, and abstinence time, and accounting for multiple semen samples (mean = 2, min-max = 1-9) in analyses for semen parameters. MAIN RESULTS AND THE ROLE OF CHANCE: Men had a median (interquartile range) age of 36 (33, 39) years and were predominantly Caucasian (87%). Of the men who completed the survey, 12% reported often lifting or moving heavy objects at work, 6% reported heavy physical exertion at work, and 9% reported evening or rotating shifts. Men who reported often lifting or moving heavy objects at work had 46% higher sperm concentrations (P = 0.01) and 44% higher total counts (P = 0.01) compared with men who reported never lifting or moving heavy objects at work. Similar results were found for men working in rotating shifts compared to those in day shifts, as well as for men involved in heavy levels of physical exertion compared to those with light levels at work. We also found that men involved in heavy/moderate levels of physical exertion at work had higher circulating testosterone concentrations compared to those with lighter exertion (adjusted means of 515 and 427 ng/dl, respectively, P = 0.08), and men who often moved/lifted heavy objects at work had higher estradiol concentrations, compared to those who never did (adjusted means of 36.8 and 27.1 pg/ml, respectively, P = 0.07). Men working evening/rotating shifts had 24% higher testosterone (P = 0.04) and 45% higher estradiol concentrations (P = 0.01), compared to men working day shifts. No associations were observed for ejaculated volume, total motility, morphologically normal sperm, or serum FSH and LH concentrations. LIMITATIONS, REASONS FOR CAUTION: Due to our study design which recruited men from couples seeking fertility treatment, it may not be possible to generalize our findings to men from the general population. Also, as is the case of all studies based on self-reported questionnaires, measurement error and misclassification of the exposure are potential concerns. WIDER IMPLICATIONS OF THE FINDINGS: Physically demanding jobs and rotating or evening shift occupations may be associated with higher testicular function in men measured as higher sperm concentrations and counts as well as higher serum testosterone and estradiol levels. Confirmation of these findings in other non-fertility clinic study populations is warranted. STUDY FUNDING/COMPETING INTEREST(S): NIH grants R01ES022955, R01ES009718, R01ES033651, and R01ES000002 from the National Institute of Environmental Health Sciences (NIEHS) and Legacy, Inc. R.A.G. works part time for Legacy, Inc., which provided funds to perform this analysis. There are no other conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

A science-based agenda for health-protective chemical assessments and decisions: overview and consensus statement.

The manufacture and production of industrial chemicals continues to increase, with hundreds of thousands of chemicals and chemical mixtures used worldwide, leading to widespread population exposures and resultant health impacts. Low-wealth communities and communities of color often bear disproportionate burdens of exposure and impact; all compounded by regulatory delays to the detriment of public health. Multiple authoritative bodies and scientific consensus groups have called for actions to prevent harmful exposures via improved policy approaches. We worked across multiple disciplines to develop consensus recommendations for health-protective, scientific approaches to reduce harmful chemical exposures, which can be applied to current US policies governing industrial chemicals and environmental pollutants. This consensus identifies five principles and scientific recommendations for improving how agencies like the US Environmental Protection Agency (EPA) approach and conduct hazard and risk assessment and risk management analyses: (1) the financial burden of data generation for any given chemical on (or to be introduced to) the market should be on the chemical producers that benefit from their production and use; (2) lack of data does not equate to lack of hazard, exposure, or risk; (3) populations at greater risk, including those that are more susceptible or more highly exposed, must be better identified and protected to account for their real-world risks; (4) hazard and risk assessments should not assume existence of a "safe" or "no-risk" level of chemical exposure in the diverse general population; and (5) hazard and risk assessments must evaluate and account for financial conflicts of interest in the body of evidence. While many of these recommendations focus specifically on the EPA, they are general principles for environmental health that could be adopted by any agency or entity engaged in exposure, hazard, and risk assessment. We also detail recommendations for four priority areas in companion papers (exposure assessment methods, human variability assessment, methods for quantifying non-cancer health outcomes, and a framework for defining chemical classes). These recommendations constitute key steps for improved evidence-based environmental health decision-making and public health protection.

Hormone receptor activities of complex mixtures of known and suspect chemicals in personal silicone wristband samplers worn in office buildings.

Humans are exposed to increasingly complex mixtures of hormone-disrupting chemicals from a variety of sources, yet, traditional research methods only evaluate a small number of chemicals at a time. We aimed to advance novel methods to investigate exposures to complex chemical mixtures. Silicone wristbands were worn by 243 office workers in the USA, UK, China, and India during four work shifts. We analyzed extracts of the wristbands for: 1) 99 known (targeted) chemicals; 2) 1000+ unknown chemical features, tentatively identified through suspect screening; and 3) total hormonal activities towards estrogen (ER), androgen (AR), and thyroid hormone (TR) receptors in human cell assays. We evaluated associations of chemicals with hormonal activities using Bayesian kernel machine regression models, separately for targeted versus suspect chemicals (with detection ≥50%). Every wristband exhibited hormonal activity towards at least one receptor: 99% antagonized TR, 96% antagonized AR, and 58% agonized ER. Compared to men, women were exposed to mixtures that were more estrogenic (180% higher, adjusted for country, age, and skin oil abundance in wristband), anti-androgenic (110% higher), and complex (median 836 detected chemical features versus 780). Adjusted models showed strong associations of jointly increasing chemical concentrations with higher hormonal activities. Several targeted and suspect chemicals were important co-drivers of overall mixture effects, including chemicals used as plasticizers, fragrance, sunscreen, pesticides, and from other or unknown sources. This study highlights the role of personal care products and building microenvironments in hormone-disrupting exposures, and the substantial contribution of chemicals not often identifiable or well-understood to those exposures.

Urinary phthalate metabolite concentrations and hot flash outcomes: Longitudinal associations in the Midlife Women's Health Study.

Midlife in women is an understudied time for environmental chemical exposures and menopausal outcomes. Recent cross-sectional research links phthalates with hot flashes, but little is known regarding such associations over time. Our objective was to estimate longitudinal associations between repeated measures of urinary phthalate metabolite concentrations and hot flash outcomes in midlife women. Using data from the Midlife Women's Health Study (MWHS), a prospective longitudinal study, we fit generalized linear mixed-effects models (GLMMs) and Cox proportional hazards regression models to repeated measures over a 4-year period. Recruitment occurred in Baltimore and surrounding counties, Maryland, USA between 2006 and 2015. Participants were premenopausal/perimenopausal women (n = 744) aged 45-54 years, who were not pregnant, not taking menopausal symptom medication or oral contraceptives, did not have hysterectomy/oophorectomy, and irrespective of hot flash experience. Baseline mean (SD) age was 48.4 (2.45), and 65% were premenopausal. Main outcome measures included adjusted odds ratios (ORs) for 4 self-reported hot flash outcomes (ever experienced, past 30 days experience, weekly/daily, and moderate/severe), and hazard ratios (HRs) for incident hot flashes. We observed mostly increased odds of certain hot flash outcomes with higher concentrations of metabolites of di (2-ethylhexyl) phthalate (DEHP), monoisobutyl phthalate (MiBP), and a molar summary measure of plasticizer phthalate metabolites (DEHP metabolites, mono-(3-carboxypropyl) phthalate (MCPP), monobenzyl phthalate (MBzP)). Some associations between exposures and outcomes indicated decreased odds. In conclusion, phthalate metabolites were associated with certain hot flash outcomes in midlife women. Midlife may be a sensitive period for higher phthalate metabolite concentrations with respect to menopausal symptoms.

Abnormal uterine bleeding patterns determined through menstrual tracking among participants in the Apple Women's Health Study.

BACKGROUND: Use of menstrual tracking data to understand abnormal bleeding patterns has been limited because of lack of incorporation of key demographic and health characteristics and confirmation of menstrual tracking accuracy. OBJECTIVE: This study aimed to identify abnormal uterine bleeding patterns and their prevalence and confirm existing and expected associations between abnormal uterine bleeding patterns, demographics, and medical conditions. STUDY DESIGN: Apple Women's Health Study participants from November 2019 through July 2021 who contributed menstrual tracking data and did not report pregnancy, lactation, use of hormones, or menopause were included in the analysis. Four abnormal uterine bleeding patterns were evaluated: irregular menses, infrequent menses, prolonged menses, and irregular intermenstrual bleeding (spotting). Monthly tracking confirmation using survey responses was used to exclude inaccurate or incomplete digital records. We investigated the prevalence of abnormal uterine bleeding stratified by demographic characteristics and used logistic regression to evaluate the relationship of abnormal uterine bleeding to a number of self-reported medical conditions. RESULTS: There were 18,875 participants who met inclusion criteria, with a mean age of 33 (standard deviation, 8.2) years, mean body mass index of 29.3 (standard deviation, 8.0), and with 68.9% (95% confidence interval, 68.2-69.5) identifying as White, non-Hispanic. Abnormal uterine bleeding was found in 16.4% of participants (n=3103; 95% confidence interval, 15.9-17.0) after accurate tracking was confirmed; 2.9% had irregular menses (95% confidence interval, 2.7-3.1), 8.4% had infrequent menses (95% confidence interval, 8.0-8.8), 2.3% had prolonged menses (95% confidence interval, 2.1-2.5), and 6.1% had spotting (95% confidence interval, 5.7-6.4). Black participants had 33% higher prevalence (prevalence ratio, 1.33; 95% confidence interval, 1.09-1.61) of infrequent menses compared with White, non-Hispanic participants after controlling for age and body mass index. The prevalence of infrequent menses was increased in class 1, 2, and 3 obesity (class 1: body mass index, 30-34.9; prevalence ratio, 1.31; 95% confidence interval, 1.13-1.52; class 2: body mass index, 35-39.9; prevalence ratio, 1.25; 95% confidence interval, 1.05-1.49; class 3: body mass index, >40; prevalence ratio, 1.51; 95% confidence interval, 1.21-1.88) after controlling for age and race/ethnicity. Those with class 3 obesity had 18% higher prevalence of abnormal uterine bleeding compared with healthy-weight participants (prevalence ratio, 1.18; 95% confidence interval, 1.02-1.38). Participants with polycystic ovary syndrome had 19% higher prevalence of abnormal uterine bleeding compared with participants without this condition (prevalence ratio, 1.19; 95% confidence interval, 1.08-1.31). Participants with hyperthyroidism (prevalence ratio, 1.34; 95% confidence interval, 1.13-1.59) and hypothyroidism (prevalence ratio, 1.17; 95% confidence interval, 1.05-1.31) had a higher prevalence of abnormal uterine bleeding, as did those reporting endometriosis (prevalence ratio, 1.28; 95% confidence interval, 1.12-1.45), cervical dysplasia (prevalence ratio, 1.20; 95% confidence interval, 1.03-1.39), and fibroids (prevalence ratio, 1.14; 95% confidence interval, 1.00-1.30). CONCLUSION: In this cohort, abnormal uterine bleeding was present in 16.4% of those with confirmed menstrual tracking. Black or obese participants had increased prevalence of abnormal uterine bleeding. Participants reporting conditions such as polycystic ovary syndrome, thyroid disease, endometriosis, and cervical dysplasia had a higher prevalence of abnormal uterine bleeding.

Preconception Stress and Pregnancy Serum Glucose Levels Among Women Attending a Fertility Center.

Context: The association between women's stress and pregnancy glucose levels remain unclear, specifically when considering the preconception period as a sensitive window of exposure. Objective: We investigated whether preconception perceived stress was associated with glucose levels during pregnancy among women attending a fertility center (2004-2019). Methods: Before conception, women completed a psychological stress survey using the short version of the validated Perceived Stress Scale 4 (PSS-4), and blood glucose was measured using a 50-gram glucose load test during late pregnancy as a part of screening for gestational diabetes. Linear and log-binomial regression models were used to assess associations of total PSS-4 scores with mean glucose levels and abnormal glucose levels ( ≥ 140 mg/dL), adjusting for age, body mass index, race, smoking, education, physical activity, primary infertility diagnosis, number of babies, and mode of conception. Results: Psychological stress was positively associated with mean abnormal glucose levels. The adjusted marginal means (95% CI) of mean glucose levels for women in the first, second, and third tertiles of psychological stress were 115 (110, 119), 119 (115, 123), and 124 (119, 128), and mg/dL, respectively (P for trend = .007). Also, women in the second and third tertiles of psychological stress had 4% and 13% higher probabilities of having abnormal glucose compared with women in the first tertile of psychological stress (P trend = .01). Conclusion: These results highlight the importance of considering preconception when evaluating the relationship between women's stress and pregnancy glucose levels.

Self-reported history of comorbidities and markers of ovarian reserve among subfertile women.

PURPOSE: To investigate whether history of comorbidities is associated with markers of ovarian reserve among subfertile women. METHODS: This observational study includes 645 women seeking fertility care at the Massachusetts General Hospital who enrolled in the Environment and Reproductive Health (EARTH) study (2005-2019). Women completed a comprehensive questionnaire including medical diagnosis of comorbidities. Ovarian reserve markers including antral follicle count (AFC), assessed by transvaginal ultrasound, and circulating serum levels of day 3 FSH and AMH, are assessed by immunoassays. We fit linear regression models to evaluate the association between history of comorbidities and markers of ovarian reserve while adjusting for confounders. RESULTS: Self-reported history of hypertension, cancer, and neurological disorders was negatively associated with AFC in unadjusted models and in adjusted models for age, smoking, physical activity, comorbidity count, and BMI. Adjusted mean AFC (95% CI) was lower among women with history of hypertension, compared to women with no self-reported history of hypertension (11.5 vs 15.6, p value 0.0001). In contrast, day 3 FSH levels were positively related to history of eating disorders in both unadjusted and adjusted models (10.8 vs. 7.43 IU/L, p value ≤ 0.0001). Self-reported history of other comorbidities was unrelated to AFC, day 3 FSH, and AMH levels. CONCLUSIONS: History of hypertension, cancer, and neurological disorders was negatively associated with AFC, and eating disorders were positively related to day 3 FSH levels. The prevention of common comorbidities among women in reproductive age may help increase women's fertility given the declining birth rates and increasing use of assisted reproductive technologies in the past years.

Maternal Serum α-Fetoprotein Levels during Pregnancy and Testicular Cancer in Male Offspring: A Cohort Study within a Danish Pregnancy Screening Registry.

Testicular cancer is believed to originate from disruptions of normal androgen-estrogen balance in-utero. α-fetoprotein (AFP) may modify fetal response to estrogens via estrogen interaction. In a cohort study, we investigated the association between circulating maternal pregnancy AFP and testicular cancer risk in offspring. Of the 56,709 live-born males from a pregnancy screening registry in 1980-1995, our study included 50,519 singleton males with available second trimester blood samples from their mothers and complete covariate ascertainment. Testicular cancer diagnoses and covariate data were obtained from nationwide Danish health registries. Cox regression and Kaplan-Meier analyses estimated the prospective risk of testicular cancer (all, seminoma, nonseminoma) by AFP multiples of the median. During follow-up, 163 (0.3%) of the included males developed testicular cancer, of which 89 (54.6%) were nonseminomas. Maternal serum AFP levels greater than/equal to the median were associated with a relative risk of testicular cancer close to unity (RR 1.04, 95% CI 0.76; 1.41) compared to AFP below the median. Associations differed by type of testicular cancer (RRseminoma 0.81, 95% CI 0.51; 1.29, RRnonseminoma 1.31, 95% CI 0.85; 2.02). On balance, our findings do not support that serum AFP in pregnancy can be used as a predictor of testicular cancer in offspring.