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Background: Interest in menopausal symptoms in general and hot flushes (HFs) in particular has grown in recent years. This is mostly due to increased awareness and the vast impact these symptoms have on women's lives. Despite the high prevalence of women who experience HFs, a definitive etiology for HFs is yet to be found. Our objective was to review the current literature dealing with associated factors for experiencing HFs and to provide a synthesized overview on this common and often debilitating condition. Methods: We systematically searched the English-language literature in the PubMed database using relevant key words and included only those articles that contained information on associated factors for HFs in generally healthy midlife women. Results: Both conflicting scientific results between studies documenting factors that influence HFs and the lack of validated measuring tools make it difficult to truly pinpoint associated factors for HFs. Nonetheless, we identified the following clusters of associated factors: the menopausal stages, sex steroid hormones, other endocrine agents, genetic polymorphisms, race/ethnicity, body mass index (BMI) and obesity, mood disorders, smoking, soy isoflavones and phytoestrogens, alcohol consumption, and physical activity. Conclusions: No single associated factor was consistently identified as having a major role in experiencing HFs. More resources should be directed to develop a unified study system along with multivariable analyses to get a better understanding of this condition, which often imposes a tremendous social and personal toll on the women who experience it. [ABSTRACT FROM AUTHOR]

Context: The value of exercise testing in women has been questioned.Objective: To determine the prognostic value of exercise testing in a population-based cohort of asymptomatic women followed up for 20 years.Design and Setting: Near-maximal Bruce-protocol treadmill test data from the Lipid Research Clinics Prevalence Study (1972-1976) with follow-up through 1995.Participants: A total of 2994 asymptomatic North American women, aged 30 to 80 years, without known cardiovascular disease.Main Outcome Measures: Cardiovascular and all-cause mortality.Results: There were 427 (14%) deaths during 20 years of follow-up, of which 147 were due to cardiovascular causes. Low exercise capacity, low heart rate recovery (HRR), and not achieving target heart rate were independently associated with increased all-cause and cardiovascular mortality. There was no increased cardiovascular death risk for exercise-induced ST-segment depression (age-adjusted hazard ratio, 1.02; 95% confidence interval [CI], 0.57-1.80; P =.96). The age-adjusted hazard ratio for cardiovascular death for every metabolic equivalent (MET) decrement in exercise capacity was 1.20 (95% CI, 1.18-1.30; P<.001); for every 10 beats per minute decrement in HRR, the hazard ratio was 1.36 (95% CI, 1.19-1.55; P<.001). After adjusting for multiple other risk factors, women who were below the median for both exercise capacity and HRR had a 3.5-fold increased risk of cardiovascular death (95% CI, 1.57-7.86; P =.002) compared with those above the median for both variables. Among women with low risk Framingham scores, those with below median levels of both exercise capacity and HRR had significantly increased risk compared with women who had above median levels of these 2 exercise variables, 44.5 and 3.5 cardiovascular deaths per 10 000 person-years, respectively (hazard ratio for cardiovascular death, 12.93; 95% CI, 5.62-29.73; P<.001).Conclusion: The prognostic value of exercise testing in asymptomatic women derives not from electrocardiographic ischemia but from fitness-related variables. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To describe long-term mortality trends by ethnicity, sex, and age for selected cancers and to assess the effect of age-adjustment using different standard populations on rate ratios and rate differences comparing black to white cancer mortality. DESIGN: Mortality rates for selected cancers were obtained from published reports of the Vital Statistics of the United States (1950-1992). All ethnic- and sex-specific cancer rates were directly age-adjusted to the total 1970 US standard population and to a subset of the 1970 US standard population 40 years and older. RESULTS: Over a 42-year period, lung cancer mortality increased in all population subgroups. Colorectal cancer mortality declined in whites, but increased in blacks. Prostate cancer mortality increased slightly in white men, but dramatically increased in black men. Breast cancer mortality stabilized in white women, but increased markedly in black women. Uterine cancer mortality declined for both ethnicities, while ovarian cancer mortality rates increased for both ethnicities. As expected, the ratios of the age-adjusted cancer mortality rates comparing blacks to whites were the same regardless of the age structure used as the standard population. In contrast, the differences in the age-adjusted rates between blacks and whites were greater when the age-truncated standard population was used. CONCLUSIONS: There are unexplained ethnic differences in the long-term mortality trends of selected cancers. Of particular concern are the increasing death rates in black individuals from colorectal, prostate, breast, and ovarian cancers. Since almost all deaths from these cancers occur in persons over 40, age-adjustment using an age-truncated standard population that includes only those age groups at risk should be considered, particularly when the question to be addressed is one dealing with the impact of a characteristic, such as ethnicity or sex, on mortality risk. [ABSTRACT FROM AUTHOR]

Objective: We sought to determine if genetic polymorphisms in the aryl hydrocarbon receptor signaling pathway are associated with menopausal hot flashes via hormone levels.Study Design: Women (n = 639) aged 45-54 years completed a study survey and provided blood for genetic and hormone analyses. The associations were analyzed using multivariable logistic regression and generalized linear models.Results: Women carrying CYP1B1 (rs1800440) GG genotype had 3-fold greater odds of experiencing hot flashes for ≥1 year compared to the AA genotype (adjusted odds ratio [OR], 3.05; 95% confidence interval [CI], 1.12-8.25). Adding serum estradiol concentrations to the confounder-adjusted model resulted in a nonsignificant association (adjusted OR, 2.59; 95% CI, 0.91-7.18). Carriers of both CYP1B1 (rs1800440) G and CYP1B1 (rs1058636) G alleles had higher odds of experiencing hot flashes for ≥1 year compared to women homozygous for the major alleles (adjusted OR, 1.77; 95% CI, 1.06-2.96), even after adjustment for serum estradiol.Conclusion: CYP1B1 is associated with menopausal hot flashes via pathways that may involve changes in serum estradiol concentration. [ABSTRACT FROM AUTHOR]

Neonicotinoids are some of the most widely used insecticides in the world because they broadly target chewing and sucking insects. Neonicotinoids are used in commercial agricultural systems, sold for use in home gardens, and found in veterinary pharmaceuticals in the form of flea and tick preventatives for companion animals. They are also used as crop seed treatments and spread throughout crops as they mature. As a result, humans, wildlife, livestock, and pets are routinely exposed to neonicotinoids through consumption of contaminated food and water as well as through use of some veterinary pharmaceuticals. Although several studies indicate that neonicotinoid exposure causes genotoxicity, neurotoxicity, hepatotoxicity, and immunotoxicity in some non-target species, the impact of neonicotinoid pesticides on the male and female reproductive systems in mammals is largely understudied. This review summarizes current insights on the impact of common neonicotinoid pesticides such as acetamiprid, clothianidin, imidacloprid, and thiacloprid on male and female reproductive health in mammals. The review also summarizes the impacts of exposure to mixtures of neonicotinoids on reproductive endpoints. In addition, this review highlights where gaps in research on neonicotinoid pesticides and reproductive health exist so that future studies can be designed to fill current gaps in knowledge., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Phthalate monoesters have been identified as endocrine disruptors in a variety of models, yet understanding of their exact mechanisms of action and molecular targets in cells remains incomplete. Here, we set to determine whether epidemiologically relevant mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) can affect biological processes by altering cell plasma membrane fluidity or formation of cell-cell contacts. As a model system, we chose endometrial stromal cell lines, one of which was previously used in a transcriptomic study with MEHHP or MEHHP-containing mixtures. A short-term exposure (1 h) of membrane preparations to endocrine disruptors was sufficient to induce changes in membrane fluidity/rigidity, whereas different mixtures showed different effects at various depths of the bilayer. A longer exposure (96 h) affected the ability of cells to form spheroids and highlighted issues with membrane integrity in loosely assembled spheroids. Finally, in spheroids assembled from T-HESC cells, MEHHP interfered with the formation of cell-cell contacts as indicated by the immunostaining of zonula occludens 1 protein. Overall, this study emphasized the need to consider plasma membrane, membrane-bound organelles, and secretory vesicles as possible biological targets of endocrine disruptors and offered an explanation for a multitude of endocrine disruptor roles documented earlier., 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 © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In Brief: This review article highlights the associations between endocrine-disrupting chemicals, reproductive aging, and menopause. Collectively, the current literature indicates that phthalates, bisphenols, parabens, per- and poly-fluoroalkyl substances, polychlorinated biphenyls, dioxins, and pesticides are associated with reproductive aging in women and animal models., Abstract: Menopause marks the end of a woman's reproductive lifetime and can have a significant effect on a woman's quality of life. Menopause naturally occurs at 51 years of age on average, but recent literature suggests that endocrine-disrupting chemicals (EDCs) in our environment can accelerate reproductive aging, causing women to reach menopause at earlier ages. This is concerning as menopause can significantly alter a woman's quality of life and is associated with increased risks of conditions such as depression, osteoporosis, and cardiovascular disease. EDC exposures have also been associated with more intense menopausal symptoms, making the menopausal transition more difficult for some women. This review highlights the associations between EDC exposure, early menopause, and reproductive aging, using both epidemiological and experimental studies.

Phthalates, synthetic chemicals widely utilized as plasticizers and stabilizers in various consumer products, present a significant concern due to their persistent presence in daily human life. While past research predominantly focused on individual phthalates, real-life human exposure typically encompasses complex mixtures of these compounds. The cumulative effects of prolonged exposure to phthalate mixtures on uterine health remain poorly understood. To address this knowledge gap, we conducted studies utilizing adult female mice exposed to a phthalate mixture for 6 and 12 months through ad libitum chow consumption. We previously reported that continuous exposure to this phthalate mixture for 6 months led to uterine fibrosis. In this study, we show that the exposure, when continued beyond 6 months to 1 year, caused fibrotic uteri to display hyperplasia with a significant increase in gland to stroma ratio. Endometrial hyperplasia is commonly caused by unopposed estrogen action, which promotes increased expression of pro-inflammatory cytokines and chemokines and proliferation of the endometrial epithelial cells. Indeed, RNA sequencing analysis revealed a marked upregulation of several estrogen-regulated genes, Wnt ligands that are involved in oncogenic pathways, as well as chemokines, in phthalate-exposed uterine tissues. Consequently, the exposed uteri exhibited increased proliferation of endometrial epithelial cells, and a heightened inflammatory response indicated by extensive homing of macrophages. Further studies revealed a marked enhancement of the Wnt/β-Catenin signaling pathway, potentially contributing to the development of endometrial hyperplasia. Collectively, this study underscores the significance of understanding the exposure to environmental factors in the pathogenesis of endometrial disorders.

Phthalates are used as plasticizers and solvents in consumer products. Virtually 100% of the US population has measurable exposure levels to phthalates, however, the mechanisms by which prenatal exposure to phthalate mixtures affects reproductive health in the offspring remain unclear. Thus, this study tested the hypothesis that prenatal exposure to an environmentally relevant phthalate mixture promotes inflammation in F1 ovarian tissue. Pregnant CD-1 dams were dosed orally with vehicle control (corn oil) or phthalate mixture (20 μg/kg/d, 200 μg/kg/d, 200 mg/kg/d, 500 mg/kg/d). Pregnant dams delivered pups naturally and ovaries and sera from the F1 females were collected at postnatal day (PND) 21, PND 60, 3 mo, and 6 mo. Sera were used to measure levels of C-reactive protein (CRP). Ovaries and sera were used for cytokine array analysis. RNA was isolated from F1 ovaries and used to quantify expression of selected cytokine genes. Prenatal exposure to the mixture significantly increased the levels of CRP at 200 µg/kg/d on PND 21 compared with controls. The mixture altered 6 immune factors in sera at PND 21 and 33 immune factors in the ovary and sera at 6 mo compared with controls. The mixture increased ovarian expression of cytokines at PND 21 and decreased ovarian expression of cytokines at 6 mo compared with controls. These data suggest that prenatal exposure to a phthalate mixture interferes with the immune response in F1 female mice long after initial exposure., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Phthalates are endocrine disrupting chemicals (EDCs) found in common consumer products such as soft plastics and cosmetics. Although the knowledge regarding the adverse effects of phthalates on female fertility are accumulating, information on the hormone sensitive endometrium is still scarce. Here, we studied the effects of phthalates on endometrial cell proliferation and gene expression. Human endometrial primary epithelial and stromal cells were isolated from healthy fertile-aged women (n=3), and were compared to endometrial cell lines T-HESC and Ishikawa. Three different epidemiologically relevant phthalate mixtures were used, defined by urine samples in the Midlife Women Health Study (MWHS) cohort. Mono (2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) was used as a single phthalate control. Cells were harvested for proliferation testing and transcriptomic analyses after 24 h exposure. Even though all cell models responded differently to the phthalate exposures, many overlapping differentially expressed genes (DEGs, FDR<0.1), related to cell adhesion, cytoskeleton and mitochondria were found in all cell types. The qPCR analysis confirmed that MEHHP significantly affected cell adhesion gene vinculin (VCL) and NADH:ubiquinone oxidoreductase subunit B7 (NDUFB7), important for oxidative phosphorylation. Benchmark dose modelling showed that MEHHP had significant concentration-dependent effects on cytoskeleton gene actin-beta (ACTB). In conclusion, short 24 h phthalate exposures significantly altered gene expression cell-specifically in human endometrial cells, with six shared DEGs. The mixture effects were similar to those of MEHHP, suggesting MEHHP could be the main driver in the mixture. Impact of phthalate exposures on endometrial functions including receptivity should be addressed., 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 © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Neonicotinoids are the most widely used insecticides in the world. They are synthetic nicotine derivatives that act as nicotinic acetylcholine receptor agonists. Although parent neonicotinoids have low affinity for the mammalian nicotinic acetylcholine receptor, they can be activated in the environment and the body to positively charged metabolites with high affinity for the mammalian nicotinic acetylcholine receptor. Imidacloprid, the most popular neonicotinoid, and its bioactive metabolite desnitro-imidacloprid differentially interfere with ovarian antral follicle physiology in vitro, but their effects on ovarian nicotinic acetylcholine receptor subunit expression are unknown. Furthermore, ovarian nicotinic acetylcholine receptor subtypes have yet to be characterized in the ovary. Thus, this work tested the hypothesis that ovarian follicles express nicotinic acetylcholine receptors and their expression is differentially modulated by imidacloprid and desnitro-imidacloprid in vitro. We used polymerase chain reaction, RNA in situ hybridization, and immunohistochemistry to identify and localize nicotinic acetylcholine receptor subunits (α2, 4, 5, 6, 7 and β1, 2, 4) expressed in neonatal ovaries (NO) and antral follicles. Chrnb1 was expressed equally in NO and antral follicles. Chrna2 and Chrnb2 expression was higher in antral follicles compared to NO and Chrna4, Chrna5, Chrna6, Chrna7, and Chrnb4 expression was higher in NO compared to antral follicles. The α subunits were detected throughout the ovary, especially in oocytes and granulosa cells. Imidacloprid and desnitro-imidacloprid dysregulated the expression of multiple nicotinic acetylcholine receptor subunits in NO, but only dysregulated one subunit in antral follicles. These data indicate that mammalian ovaries contain nicotinic acetylcholine receptors, and their susceptibility to imidacloprid and desnitro-imidacloprid exposure varies with the stage of follicle maturity., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Humans are widely exposed to phthalates, which are metabolized in the body and excreted in urine. Phthalate metabolites are excreted within hours of exposure, making urinary phthalate biomarker concentrations highly variable., Objective: The goal of this study was to characterize the long-term variability in phthalate biomarker concentrations in women across the midlife transition and to identify factors that may be associated with increased variability in those phthalate biomarker concentrations by analyzing longitudinal urinary phthalate metabolite data from the Midlife Women's Health Study (2006-2015)., Methods: A total of 741 women were enrolled in the study for a period of up to 4 years, during which they each provided 2-4 urine samples per year over 4 consecutive weeks that were pooled for analysis (1876 total pools). Nine phthalate metabolites were assessed individually and as molar sums representative of common compounds (all phthalates: ƩPhthalates; DEHP: ƩDEHP), exposure sources (plastics: ƩPlastic; personal care products: ƩPCP), and modes of action (anti-androgenic: ƩAA). Phthalate metabolites were analyzed by quartile using generalized linear models. In addition, the impact of explanatory variables (race, annual family income, and type of work) on phthalate quartile was examined using ordinal logistic regression models., Impact Statement: Phthalate biomarker concentrations are highly variable among midlife women over time, and annual sampling may not be sufficient to fully characterize long-term exposure., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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., 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 © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A previous study using miRNA sequencing revealed that exposure to a mixture of phthalates during pregnancy and lactation dysregulated rno-miR-184 and rno-miR-141-3p in the ventral prostate (VP) of offspring. Here, rno-miR-184 and rno-miR-141-3 expressions were obtained by RT-qPCR in the VP of F1 males as well as in F2 offspring, aiming to establish a relationship with possible oncogenic targets through in silico analyses with multigenerational approach. Additionally, some targets were measured by western blots to highlight a possible relationship between the deregulated miRNAs and some of their targets. VP samples from rats exposed to a mixture of phthalates maternally during pregnancy and lactation (GD10 to PND21-F1) and VP from offspring (F2) were examined. The phthalate mixture at both concentrations (20 μg and 200 mg/kg/day) increased the expression of both miRNAs in the F1 (PND22 and 120) and F2 (descendants of F1-treated males) prostate. Target prediction analysis revealed that both microRNAs are responsible for modulating the expression and synthesis of 40 common targets. A phthalate target association analysis and the HPA database showed an interesting relationship among these possible miRNAs modulated targets with prostate adenocarcinoma and other oncogenic processes. Western blots showed alteration in P63, P53, WNT5, and STAT3 expression, which are targeted by the miRNAs, in the VP of F1/F2 males. The data draw attention to the epigenetic modulation in the prostate of descendants exposed to phthalates and adds to one of the few currently found in the literature to point to microRNAs signature as biomarkers of exposure to plasticizers., (© 2024 Wiley Periodicals LLC.)

Prenatal exposure to Di (2-ethylhexyl) phthalate (DEHP) impairs the reproductive system and causes fertility defects in male offspring. Additionally, high-fat (HF) diet is a risk factor for reproductive disorders in males. In this study, we tested the hypothesis that prenatal exposure to a physiologically relevant dose of DEHP in conjunction with HF diet synergistically impacts reproductive function and fertility in male offspring. Female mice were fed a control or HF diet 7 days prior to mating and until their litters were weaned on postnatal day 21. Pregnant dams were exposed to DEHP or vehicle from gestational day 10.5 until birth. The male offspring's gross phenotype, sperm quality, serum hormonal levels, testicular histopathology, and testicular gene expression pattern were analyzed. Male mice born to dams exposed to DEHP + HF had smaller testes, epididymides, and shorter anogenital distance compared with those exposed to HF or DEHP alone. DEHP + HF mice had lower sperm concentration and motility compared with DEHP mice. Moreover, DEHP + HF mice had more apoptotic germ cells, fewer Leydig cells, and lower serum testosterone levels than DEHP mice. Furthermore, testicular mRNA expression of Dnmt1 and Dnmt3a was two to eight-fold higher than in DEHP mice by qPCR, suggesting that maternal HF diet and prenatal DEHP exposure additively impact gonadal function by altering the degree of DNA methylation in the testis. These results suggest that the combined exposure to DEHP and high-fat synergistically impairs reproductive function in male offspring, greater than exposure to DEHP or HF diet alone., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Important differences in health that are closely linked with social disadvantage exist within and between countries. According to the World Health Organization, life expectancy and good health continue to increase in many parts of the world, but fail to improve in other parts of the world, indicating that differences in life expectancy and health arise due to the circumstances in which people grow, live, work, and age, and the systems put in place to deal with illness. Marginalized communities experience higher rates of certain diseases and more deaths compared to the general population, indicating a profound disparity in health status. Although several factors place marginalized communities at high risk for poor health outcomes, one important factor is exposure to air pollutants. Marginalized communities and minorities are exposed to higher levels of air pollutants than the majority population. Interestingly, a link exists between air pollutant exposure and adverse reproductive outcomes, suggesting that marginalized communities may have increased reproductive disorders due to increased exposure to air pollutants compared to the general population. This review summarizes different studies showing that marginalized communities have higher exposure to air pollutants, the types of air pollutants present in our environment, and the associations between air pollution and adverse reproductive outcomes, focusing on marginalized communities.

Background: The European Food Safety Authority (EFSA) recommended lowering their estimated tolerable daily intake (TDI) for bisphenol A (BPA) 20,000-fold to 0.2  ng / kg  body weight  ( BW ) / day . BPA is an extensively studied high production volume endocrine disrupting chemical (EDC) associated with a vast array of diseases. Prior risk assessments of BPA by EFSA as well as the US Food and Drug Administration (FDA) have relied on industry-funded studies conducted under good laboratory practice protocols (GLP) requiring guideline end points and detailed record keeping, while also claiming to examine (but rejecting) thousands of published findings by academic scientists. Guideline protocols initially formalized in the mid-twentieth century are still used by many regulatory agencies. EFSA used a 21st century approach in its reassessment of BPA and conducted a transparent, but time-limited, systematic review that included both guideline and academic research. The German Federal Institute for Risk Assessment (BfR) opposed EFSA's revision of the TDI for BPA., Objectives: We identify the flaws in the assumptions that the German BfR, as well as the FDA, have used to justify maintaining the TDI for BPA at levels above what a vast amount of academic research shows to cause harm. We argue that regulatory agencies need to incorporate 21st century science into chemical hazard identifications using the CLARITY-BPA (Consortium Linking Academic and Regulatory Insights on BPA Toxicity) nonguideline academic studies in a collaborative government-academic program model., Discussion: We strongly endorse EFSA's revised TDI for BPA and support the European Commission's (EC) apparent acceptance of this updated BPA risk assessment. We discuss challenges to current chemical risk assessment assumptions about EDCs that need to be addressed by regulatory agencies to, in our opinion, become truly protective of public health. Addressing these challenges will hopefully result in BPA, and eventually other structurally similar bisphenols (called regrettable substitutions) for which there are known adverse effects, being eliminated from all food-related and many other uses in the EU and elsewhere. https://doi.org/10.1289/EHP13812.

Di(2-ethylhexyl) phthalate (DEHP) is a pervasive environmental toxicant used in the manufacturing of numerous consumer products, medical supplies, and building materials. DEHP is metabolized to mono(2-ethylhexyl) phthalate (MEHP). MEHP is an endocrine disruptor that adversely affects folliculogenesis and steroidogenesis in the ovary, but its mechanism of action is not fully understood. Thus, we tested the hypothesis that the aryl hydrocarbon receptor (AHR) plays a functional role in MEHP-mediated disruption of folliculogenesis and steroidogenesis. CD-1 mouse antral follicles were isolated and cultured with MEHP (0-400 μM) in the presence or absence of the AHR antagonist CH223191 (1 μM). MEHP treatment reduced follicle growth over a 96-h period, and this effect was partially rescued by co-culture with CH223191. MEHP exposure alone increased expression of known AHR targets, cytochrome P450 (CYP) enzymes Cyp1a1 and Cyp1b1, and this induction was blocked by CH223191. MEHP reduced media concentrations of estrone and estradiol compared to control. This effect was mitigated by co-culture with CH223191. Moreover, MEHP reduced the expression of the estrogen-sensitive genes progesterone receptor (Pgr) and luteinizing hormone/choriogonadotropin receptor (Lhcgr) and co-treatment with CH223191 blocked this effect. Collectively, these data indicate that MEHP activates the AHR to impair follicle growth and reduce estrogen production and signaling in ovarian antral follicles., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Di(2-ethylhexyl) phthalate and diisononyl phthalate are widely used as plasticizers in polyvinyl chloride products. Short-term exposures to phthalates affect hormone levels, ovarian follicle populations, and ovarian gene expression. However, limited data exist regarding the effects of long-term exposure to phthalates on reproductive functions. Thus, this study tested the hypothesis that short-term and long-term exposure to di(2-ethylhexyl) phthalate or diisononyl phthalate disrupts follicle dynamics, ovarian and pituitary gene expression, and hormone levels in female mice. Adult CD-1 female mice were exposed to vehicle, di(2-ethylhexyl) phthalate, or diisononyl phthalate (0.15 ppm, 1.5 ppm, or 1500 ppm) via the chow for 1 or 6 months. Short-term exposure to di(2-ethylhexyl) phthalate (0.15 ppm) and diisononyl phthalate (1.5 ppm) decreased serum follicle-stimulating hormone levels compared to control. Long-term exposure to di(2-ethylhexyl) phthalate and diisononyl phthalate (1500 ppm) increased the percentage of primordial follicles and decreased the percentages of preantral and antral follicles compared to control. Both phthalates increased follicle-stimulating hormone levels (di(2-ethylhexyl) phthalate at 1500 ppm; diisononyl phthalate at 1.5 ppm) and decreased luteinizing hormone levels (di(2-ethylhexyl) phthalate at 0.15 and 1.5 ppm; diisononyl phthalate at 1.5 ppm and 1500 ppm) compared to control. Furthermore, both phthalates altered the expression of pituitary gonadotropin subunit genes (Cga, Fshb, and Lhb) and a transcription factor (Nr5a1) that regulates gonadotropin synthesis. These data indicate that long-term exposure to di(2-ethylhexyl) phthalate and diisononyl phthalate alters follicle growth dynamics in the ovary and the expression of gonadotropin subunit genes in the pituitary and consequently luteinizing hormone and follicle-stimulating hormone synthesis., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Phthalates are chemicals ubiquitously used in industry. Individual phthalates have been found to adversely affect female reproduction; however, humans are exposed to a mixture of phthalates daily, primarily through ingestion. Previous studies show that exposure to an environmentally relevant mixture of phthalates (Mix) can affect female reproduction. Little research, however, has been conducted on the effects of short-term (1 month) and long-term (6 months) exposure to Mix on ovarian functions. Thus, this study tested the hypothesis that short-term and long-term exposure to Mix alters ovarian folliculogenesis, serum hormone concentrations, pituitary gene expression, and ovarian expression of genes involved in steroidogenesis, apoptosis, cell cycle regulation, and oxidative stress. Adult CD-1 female mice were exposed to vehicle control (corn oil) or Mix (0.15-1500 ppm) in the chow for 1 or 6 months. Exposure to Mix for 1 month increased the number of atretic follicles (0.15 ppm), altered ovarian gene expression (0.15 ppm, 1500 ppm), and decreased serum testosterone (1.5 ppm) compared to control. Exposure to Mix for 6 months increased serum follicle-stimulating hormone (FSH) (0.15 ppm), decreased serum luteinizing hormone (LH) (0.15 ppm, 1.5 ppm, and 1500 ppm), decreased serum estradiol (1500 ppm), altered pituitary gene expression (1500 ppm), increased the number (1500 ppm) and percentage (1.5 ppm and 1500 ppm) of primordial follicles, and decreased the percentage of preantral (1500 ppm) and antral (1.5 ppm and 1500 ppm) follicles compared to control. These data indicate that exposure to Mix can alter folliculogenesis, steroidogenesis, and gene expression in female mice., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jodi Flaws, Lori Raetzman, Romana Nowak, and TR Kumar report financial support was provided by National Institute of Health., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Phthalates are synthetic chemicals widely used as plasticizers and stabilizers in various consumer products. Because of the extensive production and use of phthalates, humans are exposed to these chemicals daily. While most studies focus on a single phthalate, humans are exposed to a mixture of phthalates on a regular basis. The impact of continuous exposure to phthalate mixture on uterus is largely unknown. Thus, we conducted studies in which adult female mice were exposed for 6 months to 0.15 ppm and 1.5 ppm of a mixture of phthalates via chow ad libitum. Our studies revealed that consumption of phthalate mixture at 0.15 ppm and 1.5 ppm for 6 months led to a significant increase in the thickness of the myometrial layer compared to control. Further investigation employing RNA-sequencing revealed an elevated transforming growth factor beta (TGF-β) signaling in the uteri of mice fed with phthalate mixture. TGF-β signaling is associated with the development of fibrosis, a consequence of excessive accumulation of extracellular matrix components, such as collagen fibers in a tissue. Consistent with this observation, we found a higher incidence of collagen deposition in uteri of mice exposed to phthalate mixture compared to unexposed controls. Second Harmonic Generation (SHG) imaging showed disorganized collagen fibers and nanoindentation indicated a local increase in uterine stiffness upon exposure to phthalate mixture. Collectively, our results demonstrate that chronic exposure to phthalate mixture can have adverse effects on uterine homeostasis., 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 Elsevier Inc. All rights reserved.)

Neonicotinoid insecticides are synthetic nicotine derivatives that have high affinity for invertebrate nicotine receptors and low affinity for mammalian nicotine receptors. However, imidacloprid (IMI), the most commonly used neonicotinoid, can be bioactivated by the liver in mammals to desnitro-imidacloprid, an intermediate metabolite that effectively binds and activates mammalian receptors. However, it is not known if other tissues such as the ovaries can metabolize IMI. Thus, the present study tested the hypothesis that ovarian antral follicles metabolize and bioactivate IMI. Antral follicles were dissected from the ovaries of CD-1 mice and cultured in media containing dimethyl sulfoxide or IMI (0.2-200 µg/ml) for 48 and 96 h. Media were subjected to liquid chromatography-mass spectrometry for detection of phase I IMI metabolites. Follicles from the cultures were used for gene expression analysis of metabolic enzymes associated with IMI metabolism. All IMI metabolites were detected at 48 and 96 h. Oxidized IMI intermediates were detected in media from cultured follicles, but not environmental controls. Reduced IMI intermediates were detected in media from cultured follicles and the environmental controls. At 48 h, IMI did not affect expression of any metabolic enzymes compared with control. At 96 h, IMI induced Cyp2e1 and Cyp4f18 compared with control. These data indicate that mouse ovarian follicles metabolize IMI and that IMI induces ovarian Cyp expression over time., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

As the second leading cause of death for cancer among men worldwide, prostate cancer (PCa) prevention and detection remain a critical challenge. One aspect of PCa research is the identification of common environmental agents that may increase the risk of initiation and progression of PCa. Endocrine disrupting chemicals (EDCs) are strong candidates for risk factors, partially because they alter essential pathways for prostate gland development and oncogenesis. Phthalates correspond to a set of commercially used plasticizers that humans are exposed to ubiquitously. Here, we show that maternal exposure to a phthalate mixture interferes with the expression profile of mRNA and proteins in the ventral prostate of offspring and increases the susceptibility to prostate adenocarcinomas in aged animals. The data highlight Ubxn11, Aldoc, Kif5c, Tubb4a, Tubb3, Tubb2, Rab6b and Rab3b as differentially expressed targets in young and adult offspring descendants (PND22 and PND120). These phthalate-induced targets were enriched for pathways such as: dysregulation in post-translational protein modification (PTPM), cell homeostasis, HSP90 chaperone activity, gap junctions, and kinases. In addition, the Kif5c, Tubb3, Tubb2b and Tubb4a targets were enriched for impairment in cell cycle and GTPase activity. Furthermore, these targets showed strong relationships with 12 transcriptional factors (TF), which regulate the phosphorylation of eight protein kinases. The correlation of TF-kinases is associated with alterations in immune system, RAS/ErbB/VEGF/estrogen/HIF-1 signaling pathways, cellular senescence, cell cycle, autophagy, and apoptosis. Downregulation of KIF5C, TUBB3 and RAB6B targets is associated with poor prognosis in patients diagnosed with adenocarcinoma. Collectively, this integrative investigation establishes the post-transcriptional mechanisms in the prostate that are modulated by maternal exposure to phthalate mixture during gestation and lactation., 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.)

Polychlorinated biphenyls (PCBs) were used in industrial applications until they were banned in the 1970s, but they still persist in the environment. Little is known about the long-term effects of exposure to PCB mixtures on the rat ovary during critical developmental periods. Thus, this study tested whether prenatal and postnatal exposures to PCBs affect follicle numbers and gene expression in the ovaries of F1 offspring. Sprague-Dawley rats were treated with vehicle or Aroclor 1221 (A1221) at 1 mg/kg/day during embryonic days 8-18 and/or postnatal days (PND) 1-21. Ovaries from F1 rats were collected for assessment of follicle numbers and differential expression of estrogen receptor 1 (Esr1), estrogen receptor 2 (Esr2), androgen receptor (Ar), progesterone receptor (Pgr), and Ki-67 (Ki67) at PNDs 8, 32, and 60. Sera were collected for measurement of estradiol concentrations. Prenatal exposure to A1221 significantly decreased the number of primordial follicles and the total number of follicles at PND 32 compared to control. Postnatal PCB exposure borderline increased Ki67 gene expression and significantly increased Ki67 protein levels (PND 60) compared to control. Combined prenatal and postnatal PCB exposure borderline decreased Ar expression (PND 8) compared to control. However, PCB exposure did not significantly affect the expression of Pgr, Esr1, and Esr2 or serum estradiol concentrations compared to control at any time point. In conclusion, these data suggest that PCB exposure affects follicle numbers and levels of the proliferation marker Ki67, but it does not affect expression of some sex steroid hormone receptors in the rat ovary., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Jodi Flaws reports financial support was provided by National Institute of Environmental Health Sciences. Andrea Gore reports financial support was provided by National Institute of Environmental Health Sciences. Lori Raetzman reports financial support was provided by National Institute of Environmental Health Sciences. Jodi Flaws served as an Associate Editor for Reproductive Toxicology, but her term ended January 2023. She is helping organize a special issue for the journal now., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Di-isononyl phthalate (DiNP), an endocrine-disrupting chemical, is found in numerous consumer products and human exposure to this phthalate is becoming inevitable. The impact of DiNP exposure on the establishment and maintenance of pregnancy remains largely unknown. Thus, we conducted studies in which pregnant mice were exposed to an environmentally relevant dose (20 µg/kg BW/day) of DiNP on days 1-7 of gestation, then analyzed the effects of this exposure on pregnancy outcome. Our studies revealed that exposure to DiNP during this window led to fetal loss towards the end of gestation. Further studies showed that, although embryos were able to attach to the uterus, implantation sites in DiNP-exposed uteri exhibited impaired differentiation of stromal cells to decidual cells and an underdeveloped angiogenic network in the decidual bed. We also found that exposure to this phthalate has a significant effect on trophoblast differentiation and causes disorganization of the placental layers. The labyrinth was significantly reduced, resulting in compromised expression of nutrient transporters in the placentas of mice exposed to DiNP. These placental defects in DiNP-exposed females were the cause of fetal loss during the later stages of gestation., 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 Elsevier Inc. All rights reserved.)

Chemical health risk assessment is based on single chemicals, but humans and wildlife are exposed to extensive mixtures of industrial substances and pharmaceuticals. Such exposures are life-long and correlate with multiple morbidities, including infertility. How combinatorial effects of chemicals should be handled in hazard characterization and risk assessment are open questions. Further, test systems are missing for several relevant health outcomes including reproductive health and fertility in women. Here, our aim was to screen multiple ovarian cell models for phthalate induced effects to identify biomarkers of exposure. We used an epidemiological cohort study to define different phthalate mixtures for in vitro testing. The mixtures were then tested in five cell models representing ovarian granulosa or stromal cells, namely COV434, KGN, primary human granulosa cells, primary mouse granulosa cells, and primary human ovarian stromal cells. Exposures at epidemiologically relevant levels did not markedly elicit cytotoxicity or affect steroidogenesis in short 24-hour exposure. However, significant effects on gene expression were identified by RNA-sequencing. Altogether, the exposures changed the expression of 124 genes on the average (9-479 genes per exposure) in human cell models, without obvious concentration or mixture-dependent effects on gene numbers. The mixtures stimulated distinct changes in different cell models. Despite differences, our analyses suggest commonalities in responses towards phthalates, which forms a starting point for follow-up studies on identification and validation of candidate biomarkers that could be developed to novel assays for regulatory testing or even into clinical tests., 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 The Authors. Published by Elsevier Inc. All rights reserved.)

The use of zinc oxide nanoparticles (ZnO NP) in consumer products is increasing, raising concern about their potential toxicity to human health. Nanoparticles have endocrine disrupting effects and can induce oxidative stress, leading to biomolecule oxidation and cell dysfunction. The ovary is one of the most important endocrine organs in female reproduction. Nanoparticles accumulate in the ovary, but it is unknown whether and how exposure to these materials disrupts antral follicle functions. Thus, this study tested the hypothesis that the in vitro exposure to ZnO NPs affects the steroidogenic pathway and induces oxidative stress in ovarian antral follicles. Antral follicles from CD-1 mice were cultured with ZnO NPs (5, 10, and 15 µg/mL) for 96 h. ZnO NP exposure did not affect apoptosis and cell cycle regulators at any of the tested concentrations. ZnO NP exposure at low levels (5 µg/mL) increased aromatase levels, leading to increased estradiol levels and decreased estrogen receptor alpha ( Esr1 ) expression. ZnO NP exposure at 15 µg/mL induced an antioxidant response in the antral follicles as evidenced by changes in expression of antioxidant molecules ( Nrf2 , Cat , Sod1 , Gsr , Gpx ) and decreased levels of reactive oxygen species. Interestingly, ZnO NPs dissolve up to 50% in media and are internalized in cells as soon as 1 h after culture. In conclusion, ZnO NPs are internalized in antral follicles, leading to increased estrogen production and an antioxidant response.

Polychlorinated-biphenyls (PCBs) are industrial compounds, which were widely used in manufacturing of electrical parts and transformers. Despite being banned in 1979 due to human health concerns, they persist in the environment. In humans and experimental model systems, PCBs elicit toxicity in part by acting as endocrine-disrupting chemicals (EDCs). Aroclor 1221 (A1221) is a weakly estrogenic PCB mixture known to alter reproductive function in rodents. EDCs can impact hormone signaling at any level of the hypothalamic-pituitary-gonadal (HPG) axis, and we investigated the effects of A1221 exposure during the prenatal and postnatal developmental periods on pituitary hormone and steroid receptor expression in female rats. Examining offspring at 3 ages, postnatal day 8 (P8), P32 and P60, we found that prenatal exposure to A1221 increased P8 neonate pituitary luteinizing hormone beta (Lhb) mRNA and LHβ gonadotrope cell number while decreasing LH serum hormone concentration. No changes in pituitary hormone or hormone receptor gene expression were observed peri-puberty at P32. In reproductively mature rats at P60, we found pituitary follicle stimulating hormone beta (Fshb) mRNA levels increased by prenatal A1221 exposure with no corresponding alterations in FSH hormone or FSHβ expressing cell number. Estrogen receptor alpha (ERα) mRNA and protein levels were also increased at P60, but only following postnatal A1221 dosing. Together, these data illustrate that exposure to the PCB A1221, during critical developmental windows, alters pituitary gonadotropin hormone subunits and ERα levels in offspring at different phases of maturation, potentially impacting reproductive function in concert with other components of the HPG axis., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Lori Raetzman reports financial support was provided by National Institute of Environmental Health Sciences. Jodi Flaws reports financial support was provided by National Institute of Environmental Health Sciences., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Phthalates are endocrine-disrupting chemicals used in consumer products. Although phthalates are obesogens and affect metabolic function, it is unknown if chronic exposure for 6 months to a phthalate mixture alters adipose tissue phenotype in female mice. After vehicle or mixture exposure, white adipose tissue and brown adipose tissue (WAT and BAT) were analyzed for expression of adipogenesis, proliferation, angiogenesis, apoptosis, oxidative stress, inflammation, and collagen deposition markers. The mixture altered WAT morphology, leading to an increase in hyperplasia, blood vessel number, and expression of BAT markers (Adipoq and Fgf2) in WAT. The mixture increased the expression of the inflammatory markers, Il1β, Ccl2, and Ccl5, in WAT. The mixture also increased expression of the proapoptotic (Bax and Bcl2) and antiapoptotic (Bcl2l10) factors in WAT. The mixture increased expression of the antioxidant Gpx1 in WAT. The mixture changed BAT morphology by increasing adipocyte diameter, whitening area, and blood vessel number and decreased expression of the thermogenic markers Ucp1, Pgargc1a, and Adrb3. Furthermore, the mixture increased the expression of adipogenic markers Plin1 and Cebpa, increased mast cell number, and increased Il1β expression in BAT. The mixture also increased expression of the antioxidant markers Gpx and Nrf2 and the apoptotic marker Casp2 in BAT. Collectively, these data indicate that chronic exposure to a phthalate mixture alters WAT and BAT lipid metabolism phenotypes in female mice, leading to an apparent shift in their normal morphology. Following long-term exposure to a phthalate mixture, WAT presented BAT-like features and BAT presented WAT-like features., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Phthalates are found in plastic food containers, medical plastics, and personal care products. However, the effects of long-term phthalate exposure on female reproduction are unknown. Thus, this study investigated the effects of long-term, dietary phthalate exposure on estrous cyclicity and fertility in female mice. Adult female CD-1 mice were fed chow containing vehicle control (corn oil) or 0.15-1500 ppm of di(2-ethylhexyl) phthalate (DEHP), diisononyl phthalate (DiNP), or a mixture of phthalates (Mix) containing DEHP, DiNP, benzyl butyl phthalate, di-n-butyl phthalate, diisobutyl phthalate, and diethyl phthalate. Measurements of urinary phthalate metabolites confirmed effective delivery of phthalates. Phthalate consumption for 11 months did not affect body weight compared to control. DEHP exposure at 0.15 ppm for 3 and 5 months increased the time that the mice spent in estrus and decreased the time the mice spent in metestrus/diestrus compared to control. DiNP exposure (0.15-1500 ppm) did not significantly affect time in estrus or metestrus/diestrus compared to control. Mix exposure at 0.15 and 1500 ppm for 3 months decreased the time the mice spent in metestrus/diestrus and increased the time the mice spent in estrus compared to control. DEHP (0.15-1500 ppm) or Mix (0.15-1500 ppm) exposure did not affect fertility-related indices compared to control. However, long-term DiNP exposure at 1500 ppm significantly reduced gestational index and birth rate compared to control. These data indicate that chronic dietary exposure to phthalates alters estrous cyclicity, and long-term exposure to DiNP reduces gestational index and birth rate in mice., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Imidacloprid is a neonicotinoid pesticide used in large-scale agricultural systems, home gardens, and veterinary pharmaceuticals. Imidacloprid is a small molecule that is more water-soluble than other insecticides, increasing the likelihood of large-scale environmental accumulation and chronic exposure of non-targeted species. Imidacloprid can be converted to the bioactive metabolite desnitro-imidacloprid in the environment and body. Little is known about the mechanisms by which imidacloprid and desnitro-imidacloprid induce ovarian toxicity. Thus, we tested the hypothesis that imidacloprid and desnitro-imidacloprid differentially affect antral follicle growth and steroidogenesis in vitro. Antral follicles were dissected from the ovaries of CD-1 mice and cultured in media containing vehicle control or 0.2 µg/mL-200 µg/mL of imidacloprid or desnitro-imidacloprid for 96 h. Follicle morphology was monitored, and follicle size was measured every 24 h. At the end of the culture periods, media were used to quantify follicular hormone levels, and follicles were used for gene expression analysis of steroidogenic regulators, hormone receptors, and apoptotic factors. Imidacloprid did not affect follicle growth or morphology compared to the control. Desnitro-imidacloprid inhibited follicle growth and caused follicles to rupture in culture compared to the control. Imidacloprid increased progesterone, whereas desnitro-imidacloprid decreased testosterone and progesterone compared to the control. Desnitro-imidacloprid also changed estradiol compared to the control. At 48 h, IMI decreased the expression of Star , Cyp17a1 , Hsd17b1 , Cyp19a1 , and Esr2 and increased the expression of Cyp11a1 , Cyp19a1 , Bax , and Bcl2 compared to the control. IMI also changed the expression of Esr1 compared to the control. At 48 h, DNI decreased the expression of Cyp11a1 , Cyp17a1 , Hsd3b1 , Cyp19a1 , and Esr1 and increased the expression of Cyp11a1 , Hsd3b1 , and Bax compared to the control. At 72 h of culture, IMI significantly decreased the expression of Cyp19a1 and increased the expression of Star and Hsd17b1 compared to the control. At 72 h, DNI significantly decreased the expression of Cyp11a1 , Cyp17a1 , Hsd3b1 , and Bax and increased the expression of Esr1 and Esr2 . At 96 h, IMI decreased the expression of Hsd3b1 , Cyp19a1 , Esr1 , Bax , and Bcl2 compared to the control. At 96 h, DNI decreased the expression of Cyp17a1 , Bax , and Bcl2 and increased the expression of Cyp11a1 , Hsd3b1 , and Bax compared to the control. Together, these data suggest mouse antral follicles are targets of neonicotinoid toxicity, and the mechanisms of toxicity differ between parent compounds and metabolites.

Background/objectives: Women are ubiquitously exposed to endocrine disruptors, including phthalates. Ovarian follicles undergoing folliculogenesis (indirectly measured by ovarian volume) produce anti-Müllerian hormone (AMH) and estradiol (E2). We evaluated associations of phthalates with ovarian volume to assess whether this explained prior positive associations of phthalates with AMH and E2., Methods: Women ages 45-54 years (n = 614) had transvaginal ultrasounds of right/left ovaries to calculate mean ovarian volume. Women provided up-to-four urine and blood samples for quantifying AMH (first serum sample), E2 (all serum samples), and nine phthalate metabolites (from pooled urine, representing six parent phthalates). Multivariable linear or logistic regression models (for individual phthalate biomarkers), as well as weighted quantile sum (WQS) regression (for mixture analyses) evaluated associations of phthalate biomarkers with ovarian volume. Using cross-sectional mediation analysis, we assessed whether associations of phthalates with ovarian volume partially explained those of phthalates with AMH or E2., Results: Most women were non-Hispanic White (68%) and pre-menopausal (67%) with higher urinary phthalate metabolite concentrations than U.S. women. In single-pollutant models, 10% increases in mono(3-carboxypropyl) phthalate (MCPP) and monobenzyl phthalate (MBzP) were associated with 0.44% (95% CI: -0.02%, 0.91%) and 0.62% (95% CI: 0.02%, 1.23%) larger ovarian volumes, respectively. As a cumulative mixture, 10% increases in the phthalate mixture were associated with 2.89% larger ovarian volume (95%CI: 0.27, 5.59) with MCPP (35%) and MBzP (41%) identified as major contributors. Higher ovarian volume due to a 10% increase in MBzP (indirect effect OR: 1.004; 95% CI: 1.00, 1.01) explained 16% of the positive association between MBzP and higher AMH, whereas higher ovarian volume due to a 10% increase in MCPP (indirect effect %Δ: 0.11; 95% CI: -0.01, 0.22) explained 23% of the positive association between MCPP and E2., Conclusion: In this cross-sectional study, phthalates were associated with increased ovarian volume, with implications for midlife hormone production., 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 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Sleep disruptions are among the most common symptoms experienced during menopause and can be associated with depression, hot flashes, and fluctuating hormones. However, few studies have examined how such risk factors influence sleep in midlife women in a network-based approach that will establish the complex relationship between variables. Materials and Methods: We used a Bayesian network (BN) to examine the relationship between multiple factors known to influence sleep and depression in midlife women, including hormone concentrations, hot flashes, and menopause status among participants of the longitudinal Midlife Women's Health Study. In year 1, 762 women (45-54 years of age) answered questions regarding the frequency of insomnia, hot flashes, and depression; 389 of the same women answered similar questions at year 4. We measured serum hormones and calculated free estradiol index, free testosterone index, and ratios of estradiol:progesterone, and estradiol:testosterone. For our model, we calculated the change in frequency of insomnia, depression, and covariates (body mass index, menopause status, hot flashes at night, and present quality of life) from year 1 to 4. Results: Using a BN, we found that self-reported hot flashes at night, and no other factors, were direct predictors of self-reported insomnia in year 1. Surprisingly, we did not identify an association between hormone concentrations and self-reported insomnia. Frequency of insomnia in year 4 was only predicted by frequency of insomnia in year 1, whereas frequency of depression in year 4 was predicted by year 4 insomnia and frequency of depression in year 1. No other factors were direct predictors of insomnia or depression in our model. Conclusions: Therefore, hot flashes at night, previous insomnia, and depression are stronger predictors of how women will self-report frequency of sleep disruptions and treatment may reduce menopausal sleep complaints.

Molecular-level analyses of breast carcinogenesis benefit from vivo disease models. Estrogen receptor 1 (Esr1) and cytochrome P450 family 19 subfamily A member 1 (CYP19A1) overexpression targeted to mammary epithelial cells in genetically engineered mouse models induces largely similar rates of proliferative mammary disease in prereproductive senescent mice. Herein, with natural reproductive senescence, Esr1 overexpression compared with CYP19A1 overexpression resulted in significantly higher rates of preneoplasia and cancer. Before reproductive senescence, Esr1, but not CYP19A1, overexpressing mice are tamoxifen resistant. However, during reproductive senescence, Esr1 mice exhibited responsiveness. Both Esr1 and CYP19A1 are responsive to letrozole before and after reproductive senescence. Gene Set Enrichment Analyses of RNA-sequencing data sets showed that higher disease rates in Esr1 mice were accompanied by significantly higher expression of cell proliferation genes, including members of prognostic platforms for women with early-stage hormone receptor-positive disease. Tamoxifen and letrozole exposure induced down-regulation of these genes and resolved differences between the two models. Both Esr1 and CYP19A1 overexpression induced abnormal developmental patterns of pregnancy-like gene expression. This resolved with progression through reproductive senescence in CYP19A1 mice, but was more persistent in Esr1 mice, resolving only with tamoxifen and letrozole exposure. In summary, genetically engineered mouse models of Esr1 and CYP19A1 overexpression revealed a diversion of disease processes resulting from the two distinct molecular pathophysiological mammary gland-targeted intrusions into estrogen signaling during reproductive senescence., (Copyright © 2023 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Age is a risk factor for human estrogen receptor-positive breast cancer, with highest prevalence following menopause. While transcriptome risk profiling is available for human breast cancers, it is not yet developed for prognostication for primary or secondary breast cancer development utilizing at-risk breast tissue. Both estrogen receptor α (ER) and aromatase overexpression have been linked to human breast cancer. Herein, conditional genetically engineered mouse models of estrogen receptor 1 (Esr1) and cytochrome P450 family 19 subfamily A member 1 (CYP19A1) were used to show that induction of Esr1 overexpression just before or with reproductive senescence and maintained through age 30 months resulted in significantly higher prevalence of estrogen receptor-positive adenocarcinomas than CYP19A1 overexpression. All adenocarcinomas tested showed high percentages of ER +  cells. Mammary cancer development was preceded by a persistent proliferative transcriptome risk signature initiated within 1 week of transgene induction that showed parallels to the Prosigna/Prediction Analysis of Microarray 50 human prognostic signature for early-stage human ER +  breast cancer. CYP19A1 mice also developed ER +  mammary cancers, but histology was more divided between adenocarcinoma and adenosquamous, with one ER - adenocarcinoma. Results demonstrate that, like humans, generation of ER +  adenocarcinoma in mice was facilitated by aging mice past the age of reproductive senescence. Esr1 overexpression was associated with a proliferative estrogen pathway-linked signature that preceded appearance of ER +  mammary adenocarcinomas., (Copyright © 2023 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

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., 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., (Published by Elsevier Inc.)

Endocrine disrupting chemicals (EDCs) are exogenous substances that alter the endocrine function of an organism, to result in adverse effects on growth and development, metabolism, and reproductive function. The kidney is one of the most important organs in the urinary system and an accumulation point. Studies have shown that EDCs can cause proteinuria, affect glomeruli and renal tubules, and even lead to diabetes and renal fibrosis in animal and human studies. In this review, we discuss renal accumulation of select EDCs such as dioxins, per- and polyfluoroalkyl substances (PFAS), bisphenol A (BPA), and phthalates, and delineate how exposures to such EDCs cause renal lesions and diseases, including cancer. The regulation of typical EDCs with specific target genes and the activation of related pathways are summarized.

Di(2-ethylhexyl) phthalate (DEHP) is a large-molecular-weight phthalate added to plastics to impart versatile properties. DEHP can be found in medical equipment and devices, food containers, building materials, and children's toys. Although DEHP exposure occurs most commonly by ingesting contaminated foods in the majority of the population, its effects on the gastrointestinal tract have not been well studied. Therefore, we analyzed the effects of subchronic exposure to DEHP on the ileum and colon morphology, gene expression, and immune microenvironment. Adult C57BL/6 female mice were orally dosed with corn oil (control, n = 7) or DEHP (0.02, 0.2, or 30 mg/kg, n = 7/treatment dose) for 30-34 days. Mice were euthanized during diestrus, and colon and ileum tissues were collected for RT-qPCR and immunohistochemistry. Subchronic DEHP exposure in the ileum altered the expression of several immune-mediating factors (Muc1, Lyz1, Cldn1) and cell viability factors (Bcl2 and Aifm1). Similarly, DEHP exposure in the colon impacted the gene expression of factors involved in mediating immune responses (Muc3a, Zo2, Ocln, Il6, and Il17a); and also altered the expression of cell viability factors (Ki67, Bcl2, Cdk4, and Aifm1) as well as a specialized epithelial cell marker (Vil1). Immunohistochemical analysis of the ileum showed DEHP increased expression of VIL1, CLDN1, and TNF and decreased number of T-cells in the villi. Histological analysis of the colon showed DEHP altered morphology and reduced cell proliferation. Moreover, in the colon, DEHP increased the expression of MUC2, MUC1, VIL1, CLDN1, and TNF. DEHP also increased the number of T-cells and Type 2 immune cells in the colon. These data suggest that subchronic DEHP exposure differentially affects the ileum and colon and alters colonic morphology and the intestinal immune microenvironment. These results have important implications for understanding the effects of DEHP on the gastrointestinal system., 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 © 2022 Elsevier Ltd. All rights reserved.)