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STUDY QUESTION: Are brain-derived neurotrophic factor (BDNF) and its receptors, NTRK2, NGFR and SORT 1, regulated by ovarian steroids in the uterus? SUMMARY ANSWER: BDNF and its low affinity receptor, nerve growth factor receptor (NGFR), are regulated by estradiol in the uterus. WHAT IS KNOWN ALREADY: Recent studies have revealed a central role for neurotrophins in placental development, endometrial stem cell neurogenesis, endometrial carcinoma and endometriosis. Complex signaling pathways involving BDNF and its receptors are regulated by ovarian hormones in the brain, however their expression and regulation in the uterus is poorly defined. STUDY DESIGN, SIZE, DURATION: This experimental animal study involved a total of 80 mice. PARTICIPANTS/MATERIALS, SETTING, METHODS: Female C57BL/6 mice (n = 50) were monitored daily for estrous cycle stage, and uterine horns were collected. A second group of mice (n = 30) were ovariectomized and given estradiol, progesterone, estradiol + progesterone, or saline for 4 days. Uterine expression of BDNF and its receptors were quantified by real-time PCR and western blot, and localized using immunohistochemistry. MAIN RESULTS ANDTHE ROLE OF CHANCE: During the estrous cycle, expression of BDNF, NTRK2 and SORT1 remained constant, while NGFR declined 11-fold from pro-estrus through to diestrus (P = 0.005). In ovariectomized mice, estradiol treatment increased uterine expression of mature BDNF greater than 6-fold (P = 0.013, 25 kDa; P = 0.003, 27 kDa), pro-BDNF 5-fold (P = 0.041, 37 kDa band; P = 0.046, 40 kDa band), and NGFR 5-fold (P, 0.001) when compared with other treatments. NTRK2 and SORT 1 were unaffected by ovarian hormones. NGFR was primarily localized in epithelial cells in mice in diestrus or in ovariectomized mice treated with progesterone (P ≤ 0.001; P≤0.00l, respectively). In contrast, NGFR switched to a stromal localization in ovariectomized mice administered estradiol (P = 0.002). LIMITATIONS, REASONS FOR CAUTION: This study was performed in one only species. WIDER IMPLICATIONS OF THE FINDINGS: Results of this study demonstrate the uterine regulation of BDNF and NGFR by estradiol, and highlight the striking difference between hormone exposure during the estrous cycle and daily estradiol exposure after ovariectomy on neurotrophin expression in the uterus. The results also show the spatial regulation of NGFR in the uterus in response to ovarian hormones. Sustained estrogen exposure, as seen in estrogen-dependent disease, may alter the delicate neurotrophin balance and inappropriately activate potent BDNF-NTRK2 pathways which are capable of contributing to endometrial pathology. STUDY FUNDING/COMPETING INTERESTS: This study was supported by the Canadian Institutes of Health Research (CIHR) (W.G.F.), a NSERC Discovery Grant (W.G.F.), and a Vanier Canada Graduate Scholarship-CIHR(J.M.W.).J.M.W. is a member of the CIHR sponsored Reproduction and Early Development in Health training program. The authors declare no conflicts of interest. [ABSTRACT FROM AUTHOR]

The eutopic secretory phase endometrium in endometriosis overproduces and releases a soluble immunosuppressive CD200 molecule (CD200L) and is populated by stromal cells that contain a truncated CD200 (CD200S) that promotes a proinflammatory environment. The CD200S+ cell population persists when pregnancy occurs and are abundant in the early pregnancy decidua of women with missed abortion. In the present study, CD200S+, CD56+, and CD68+ cells were enumerated in formalin-fixed paraffin-embedded tissue sections from women with endometriosis and non-endometriosis controls. CD200S+ cells were more numerous than CD68+ macrophages and were similar in number and location to CD56bright endometrial NK cells. In some endometria, there was an additional population of CD200S- CD56+ NK cells. In ectopic endometrial peritoneal deposits and in ectopic myometrial deposits (adenomyosis), CD200S+ cells were less frequent, consistent with the known paucity of CD56+ NK cells in sites of ectopic deposits. CD200S+ cell frequency was greater in stroma surrounding the smaller ectopic cystic deposits. Dual immunofluorescent antibody staining confirmed CD200S+ cells were CD56+ NK cells. CD200S+ NK cell frequency may be greater in endometriosis patients' endometrium and may affect embryo survival in early pregnancy. In our opinion, regulation of alternative splicing that results in CD200S rather than CD200L may provide new diagnostic and therapeutic options for women with endometriosis. • A subpopulation of stromal leukocytes in secretory phase endometrium are CD200S+. • CD200S counters immunosuppression by CD200L and may promote inflammation/rejection. • CD200S+ cells in endometrium are CD56+ uterine NK cells which persist in decidua. • CD200S + uNK cells may facilitate ectopic endometrial implantation. [ABSTRACT FROM AUTHOR]

Background: The association of DDT (dichlorodiphenyltrichloroethane) with breast cancer is controversial, but animal studies directly linking DDT to risk are lacking. Concerns with DDT reside in its environmental persistence, bioaccumulation in breast adipose tissue, and endocrine-disrupting actions. Whereas most attention has been focused on estrogenic congeners, we tested the cancer-inducing potential of the antiandrogen, p,p´-DDE [1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene], the most prevalent and persistent DDT metabolite. Objectives: We aimed to determine whether developmental exposure to p,p´-DDE stored in adipose tissue surrounding the cancer-prone mammary epithelium of MMTV-Neu mice influences tumor development. Methods: For localized delivery, Elvax 40P pellets containing p,p´-DDE were implanted into the mammary fat pads of prepubertal female mice. We compared mammary tumor development with p,p´-DDE with development in response to its estrogenic isomer, o,p´-DDE [1,1-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl) ethylene], and a mixture of both isomers. Results: p,p´-DDE implants significantly accelerated mammary tumor onset compared with vehicle Elvax implants. o,p´-DDE had similar results, but only at ? 10 months of age. Lipid-adjusted levels of p,p´-DDE in mammary adipose tissue and serum in young mice were within the ranges of human exposure, whereas concentrations in aged mice were low to undetectable. Exposure to a 2:1 ratio of p,p´-DDE:o,p´-DDE did not result in the younger latency observed with the individual isomers. Conclusions: p,p´-DDE exposure at concentrations relevant to human exposure accelerates mammary carcinogenesis in mice, possibly through hormonal and/or other actions. These data suggest that DDE exposure would promote, but not cause, mammary tumorigenesis. Developmental exposure in immature mammary tissue continues to affect tumor onset even after p,p´-DDE levels have declined. Future studies are needed to determine whether early exposure to p,p´-DDE correspondingly predisposes women to early-onset breast cancer. [ABSTRACT FROM AUTHOR]

Abstract: The objective of this study was to determine the effect of in utero and lactational exposure to nicotine on the male reproductive tract. Dams were randomly assigned to receive saline or nicotine bitartrate (1mg/kg-d s.c.) daily for two weeks prior to mating until weaning (postnatal day 21). Male offspring were sacrificed at 7 (peri-pubertal) and 26 (adult) weeks of age. Nicotine-exposure resulted in retention of spermatids after stage VIII, tubular vacuolation, degeneration of pachytene and round spermatids at stage VII in the testes; and lymphocyte infiltration, germ cell exfoliation, and hypospermia in epididymides, at 7 weeks of age. Nicotine-exposure had no effect on testis or epididymal morphology, daily sperm production, epididymal sperm reserve, sperm viability at 26 weeks of age, and circulating testosterone levels at either age examined. We conclude that maternal nicotine-exposure during pregnancy and lactation can induce transient structural changes in the testis and epididymis of male offspring. [Copyright &y& Elsevier]

Brain-derived neurotrophic factor (BDNF) is thought to play a role in follicle activation and oocyte maturation. It is postulated that BDNF and its receptor, tyrosine kinase receptor B (TrkB), may also play a role in maintaining the corpus luteum. Therefore, human granulosa lutein cells (GLC) were obtained from women undergoing ovulation induction and treated with increasing concentrations of cAMP (0, 125, 500 and 1000 µmol/l). BDNF and progesterone concentrations were quantified by enzyme-linked immunosorbent assay. cAMP treatment significantly increased progesterone output but had no effect on BDNF concentration in the spent media. However, the BDNF concentration was significantly increased in GLC lysates. To assess the expression of BDNF and TrkB in active versus regressing corpora lutea, ovaries from adult female BALBc mice (n = 4) from each day of the oestrous cycle were processed for immunohistochemistry. Two markers of luteal activity were used (3β-hydroxysteroid dehydrogenase and tenascin-X). There was a trend towards higher BDNF and TrkB H-scores in active versus regressing corpus lutea. In conclusion, intracellular BNDF concentrations were dose-dependently increased by cAMP but treatments had no effect on BDNF output. It is speculated that BDNF contributes in an autocrine manner to GLC survival in the active corpus luteum. [ABSTRACT FROM AUTHOR]

It has been shown that benzo[a]pyrene, a key component of cigarette smoke and an aryl hydrocarbon receptor (AhR) ligand, reduced growth of isolated rat follicles in vitro. However, the mechanism underlying the induced changes in folliculogenesis is unknown. This study proposed that the reported adverse effects of benzo[a]pyrene on follicle growth are mediated through AhR activation. The objective was to investigate the effect of benzo[a]pyrene with and without AhR antagonists (resveratrol or 3',4'- dimethoxyflavone (3,4-DMF)) on follicle growth, oestradiol output, anti-Mullerian hormone (AMH) concentration and cell proliferation in isolated rat follicles cultured in vitro. Benzo[a]pyrene treatment significantly inhibited follicle growth and cell proliferation at concentrations of 1.5ng/ml and higher (P<0.05), an effect attenuated by co-incubation with benzo[a]pyrene and resveratrol or 3,4-DMF. A significant decrease in oestradiol (P < 0.05) and AMH output (P < 0.001) by cultured follicles was induced by benzo[a]pyrene treatment, an effect attenuated by co-incubation with 3,4-DMF. The results suggest that the adverse effects of benzo[a]pyrene on follicle growth, steroidogenesis and AMH output are mediated through activation of the AhR. Moreover, AhR antagonists such as resveratrol and 3,4-DMF may have therapeutic benefit in protecting the ovary against the adverse effects of AhR ligands, including benzo[a]pyrene. [ABSTRACT FROM AUTHOR]

Abstract: Perfluoroalkyl compounds (PFCs) are end-stage metabolic products from industrial flourochemicals used in the manufacture of plastics, textiles, and electronics that are widely distributed in the environment. The objective of the present study was to quantify exposure to perfluorooctane sulfonate (PFOS), perfluorooctanoate (PFOA), perfluorodecanoic acid (PFDeA), perfluorohexane sulfonate (PFHxS), perfluoroheptanoic acid (PFHpA), and perfluorononanoic acid (PFNA) in serum samples collected from pregnant women and the umbilical cord at delivery. Pregnant women (n=101) presenting for second trimester ultrasound were recruited and PFC residue levels were quantified in maternal serum at 24–28 weeks of pregnancy, at delivery, and in umbilical cord blood (UCB; n=105) by liquid chromatography–mass spectrometry. Paired t-test and multiple regression analysis were performed to determine the relationship between the concentrations of each analyte at different sample collection time points. PFOA and PFOS were detectable in all serum samples analyzed including the UCB. PFOS serum levels (mean±S.D.) were significantly higher (p<0.001) in second trimester maternal serum (18.1±10.9ng/mL) than maternal serum levels at delivery (16.2±10.4ng/mL), which were higher than the levels found in UCB (7.3±5.8ng/mL; p<0.001). PFHxS was quantifiable in 46/101 (45.5%) maternal and 21/105 (20%) UCB samples with a mean concentration of 4.05±12.3 and 5.05±12.9ng/mL, respectively. There was no association between serum PFCs at any time point studied and birth weight. Taken together our data demonstrate that although there is widespread exposure to PFCs during development, these exposures do not affect birth weight. [Copyright &y& Elsevier]

Abstract: Bisphenol A (BPA) is an estrogenic contaminant that has been quantified at higher levels in the follicular fluid of women with polycystic ovarian syndrome (PCOS) compared to healthy fertile controls. However, the effect of BPA on granulosa cell function is unknown. Therefore, the objective of the present study was to quantify the effect of BPA on granulosa cell progesterone (P4) output and matrix metalloproteinase (MMP)-2, and -9 output and activity. Granulosa-lutein cells (GLCs) were collected from women undergoing oocyte retrieval in an academic in vitro fertilization (IVF) program. Granulosa-lutein cells were treated with increasing log concentrations of BPA (1–10,000ng/ml) or 17β-estradiol (E2, 272pg/ml or 1.0nM) and treatment effects on MMP-2 and -9 activity and output, cell viability and cell proliferation were measured by commercial gelatin zymography, MMP-ELISA, MTS and BrdU incorporation assays, respectively. Granulosa-lutein cells in culture secrete MMP-2 and MMP-9. Bisphenol A treatment concentration-dependently increased MMP-9 output by GLCs with a maximal effect observed at 1000ng/ml. Cell viability/proliferation was unaffected by BPA treatment at concentrations ≤100ng/ml; however, higher concentrations of BPA were cytotoxic. Progesterone output by the GLCs was unaffected by increasing BPA concentrations in the media. In conclusion, GLCs in culture secrete MMP-2 and MMP-9. At lower concentrations, compatible with human exposure levels (100–1000ng/ml), BPA stimulates GLC MMP-9 output; however, higher concentrations are cytotoxic. Our findings suggest that BPA treatment can modulate ovarian extracellular matrix stability. [Copyright &y& Elsevier]

Abstract: Clinical evidence suggests an association between galactosemia and premature ovarian failure, but the mechanism is still not fully understood. Growth differentiation factor-9 (GDF-9) is thought to be an obligatory growth factor during the gonadotropin-independent phase of folliculogenesis. The objective of this study was to examine the effects of galactose on initiation of folliculogenesis in the peripubertal interval and the connection between galactose toxicity and GDF-9 expression in the ovary. After immature Long-Evans rats (n =10) were fed a diet consisting of 20% galactose for 19 days, whole body, ovary and uterine weights were measured. Serum estradiol and progesterone concentrations were measured by radioimmunoassay. Ovarian follicles were counted by morphometric analysis and GDF-9 expression was investigated by immunohistochemistry and immunoblot assay. Galactose treatment did not affect the onset of puberty as marked by the time of vaginal opening. The galactose diet significantly decreased the number of healthy growing follicles. The results of immunoblot assay showed that both bands corresponding to propeptide and mature forms of GDF-9 decreased with the galactose diet about 90 and 70%, respectively. The results of immunohistochemical staining showed that the GDF-9 positive follicle number and the ratio of GDF-9 positive to GDF negative (primordial/non-growing) follicles significantly decreased with this high galactose diet. The present study suggests that a high galactose diet inhibits follicular development, possibly through down-regulation of GDF-9 in the rat ovary, implying that GDF-9 may be involved in galactose-related ovarian toxicity. [Copyright &y& Elsevier]

BACKGROUND: 1,1‐Dichloro‐2,2‐bis(P‐chlorophenyl)ethylene (P,P′‐DDE, DDE), a metabolite of DDT, is a persistent hormonally active environmental toxicant which has been found in human serum and follicular fluid. The objective of this study was to investigate the interaction between FSH and ppDDE on aromatase activity in primary cultures of human granulosa cells. METHODS: Granulosa cells were obtained at the time of oocyte retrieval for IVF procedures and cultured in defined medium containing FSH and environmentally relevant concentrations of DDE. Aromatase activity was measured by incubating the cells with 1β‐[3H]androstenedione and measuring the release of 3H2O. RESULTS: The granulosa cell response to FSH was highly dependent on the basal level of aromatase activity (r = –0.703, P = 0.001, n = 17) with the highest activity occurring at low basal levels of aromatase activity. Enzyme activity was significantly stimulated at 100 ng DDE/ml. A synergistic effect on aromatizing activity was observed when cells were co‐cultured with DDE and FSH. CONCLUSIONS: Concentrations of DDE similar to those present in human follicular fluid enhance basal and FSH‐stimulated granulosa cell aromatizing enzyme activity. [ABSTRACT FROM AUTHOR]

Childhood exposure to phthalates, a class of chemicals with known reproductive and developmental effects, has been hypothesized to increase the risk of obesity, but this association is not well understood in preschool children. We examined the association between urinary concentrations of phthalate metabolites and concurrently measured body mass index (BMI) and skinfolds among children between the ages of two and five years. We collected anthropometric measures and biomonitoring data on approximately 200 children enrolled in the Maternal-Infant Research on Environmental Chemicals Child Development Plus study. We measured 22 phthalate metabolites in children's urine and used the 19 metabolites detected in at least 40% of samples. Our primary outcome was BMI z-scores calculated using the World Health Organization growth standards. Skinfold z-scores were secondary outcomes. We used multivariable linear regression to evaluate the association between tertiles of phthalate concentrations and each anthropometric measure. We also used weighted quantile sum regression to identify priority exposures of concern. Our analytic sample included 189 singleton-born children with complete anthropometric data. Children with concentrations of the parent compound di-n-butyl phthalate (∑DnBP) in the third tertile had 0.475 (95% CI: 0.068, 0.883) higher BMI z-scores than those in the lower tertile. ∑DnBP was identified as a priority exposure in the weighted quantile sum regression BMI model. In this population of Canadian preschool aged children, we identified DnBP as a potential chemical of concern in regard to childhood obesity. Future research with serial phthalate measurements and anthropometric measurements in young children will help confirm these findings. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate the impact of an interleukin-1 (IL-1) antagonist anakinra (Kineret ® ) on endometriosis-related quality of life (QoL), pain, and inflammatory biomarkers., Methods: This was a single-site, randomized, double-blinded, placebo-controlled, cross-over pilot clinical study of patients recruited at an academic specialty clinic. Eligible participants were females aged 18-45 years with menstrual cycles every 24-32 days. Subjects had moderate to severe dysmenorrhea and either a surgical diagnosis of endometriosis or an endometrioma on imaging. Subjects were randomly assigned in a double-blind fashion to receive either the study drug or placebo administered as daily injections during the first 3 periods and then the alternate intervention for the next 3 periods., Results: Fifteen subjects completed the 6 menstrual cycle study. After each period, they completed the Endometriosis Health Profile-30 (EHP-30) QoL questionnaire and an assessment of dysmenorrhea using a 0-100 Visual Analogue Scale (VAS). All domains of the EHP-30 showed a trend towards improvement, with significant improvements in powerlessness (54.5 vs 63.3, p = 0.04) and self-image (58.1 vs 66.7, p = 0.03) on the study drug compared to placebo. The mean dysmenorrhea VAS also trended toward improvement with a score of 37.5 during active treatment and 42.6 with placebo (p = 0.26). No difference in menstrual cycle length was detected (29.3 days vs 27.7 days, p = 0.56). There were significant differences in multiple inflammatory biomarkers between the study drug and placebo, including BDNF, IL-1, and IL-6 among certain groups., Conclusion: With all EHP-30 domains and the dysmenorrhea VAS showing either a statistical improvement or trend towards improvement, there is justification for a larger study. As no impact on menstrual cycles was detected, anakinra may be a particularly impactful option for women desiring fertility. Additional evaluation is needed on the role of anakinra on inflammatory markers given significant reductions were identified in multiple biomarkers., Competing Interests: Dr Foster and Dr Wessels are Co-Founders of Afynia Laboratories, a medical diagnostic company, and hold several patents unrelated to the treatment of endometriosis. The other authors report no conflicts of interest in this work. An abstract of this paper was presented at the 2023 AAGL Conference as a poster with interim findings. The poster’s abstract was published in the Journal of Minimally Invasive Gynecology Supplement: https://www.sciencedirect.com/science/article/pii/S1553465023006738, (© 2024 Sullender et al.)

Endometriosis is a chronic disease associated with debilitating pain that affects many people assigned female at birth, from menarche through menopause, not just causing pain and infertility, but also negatively impacting quality of life, participation in daily activities, productivity and income. It is associated with increased incidence of obstetric and neonatal complications, depression, other chronic diseases, and substantial healthcare costs. Despite the profound negative impact of endometriosis on quality of life, current treatment options remain sub-optimal and many patients express dissatisfaction with current care. The prevailing acute-care, single-provider model in which the provider works in relative isolation and thus with limited therapeutic strategies readily available, proves inadequate for treating endometriosis. Patients would benefit from earlier diagnosis and referral to a center capable of providing a comprehensive and multi-modal management plan that utilizes a chronic care model. Often this can only be achieved through multidisciplinary teams of providers with expertise in endometriosis. Researchers need to agree on standardized core outcome measures, relevant to patients with endometriosis and the healthcare system as a whole. Only through increased education and recognition of endometriosis as a chronic disease can we achieve better treatment outcomes.

Objectives: To describe the use and perceived effectiveness of medical, surgical, and alternative therapies (e.g., diet, exercise, heat, cannabis, etc.) in managing endometriosis-associated pain in Canadians., Methods: A cross-sectional online survey was distributed via The Endometriosis Network Canada (TENC) from February to March 2021. Canadians aged 18-50 years with diagnosed or suspected endometriosis were eligible to participate., Results: A total of 434 survey responses were included, and 93.8% of respondents reported that they used at least 1 alternative therapy in the past 6 months for endometriosis-associated pain. Respondents used an array of medical (2.3/6 months), surgical (1.7/lifetime), and alternative therapies (6.9/6 months) to manage their pain, yet 61.9% of respondents did not feel it was adequately managed. The most common alternative therapies were heat, meditation/mindfulness/rest, and diet., Conclusion: Alternative therapies were commonly used by Canadians living with endometriosis to manage pain. Cannabis and heat were perceived as the most effective alternative therapies. Here, we gain a better understanding of alternative therapies that can provide an additional therapeutic avenue that clinicians and people living with endometriosis may wish to discuss and explore., (Copyright © 2022 The Society of Obstetricians and Gynaecologists of Canada/La Société des obstétriciens et gynécologues du Canada. Published by Elsevier Inc. All rights reserved.)

Endometriosis is a chronic, painful disease without a cure. Due largely to chronic pain, endometriosis can lead to significant physical, mental, relationship, and financial burdens. Within the conventional single provider model of care-in which the patient is primarily taken care of by her physician and complementary strategies based on psychology, nutrition, pain medicine, pelvic physical therapy, and so on may not be readily available in a coordinated manner-most women with endometriosis live with unresolved pain and the consequences of that pain. We therefore propose that there is an urgent need to search for alternative models of care. In the current paper, we discuss our experiences with an model of care in which we adopt a long-term, patient-focused, and multidisciplinary chronic care model for women with endometriosis. Our objective is to improve long-term clinical outcomes for women with endometriosis. For geographical areas and healthcare systems in which it is feasible, we propose consideration of this multidisciplinary model of care as an alternative to the single provider model and offer guidance for those considering establishment of such a program. We also initiate a conversation about which clinical outcomes pertaining to endometriosis are important and should be tracked to assess the efficacy and value of multidisciplinary and other endometriosis healthcare models., Competing Interests: The authors report no conflicts of interest in this work.

Exposure to environmental toxicants has been associated with ovarian dysfunction yet sensitive biomarkers of adverse effect are lacking. We previously demonstrated that cigarette smoke exposure induced decreased relative ovarian weight, increased follicle loss and granulosa cell autophagy in mice. We postulate that cigarette smoke exposure will induce changes in the epigenome that can be used to reveal potential sensitive biomarkers of ovarian toxicity. Therefore, we evaluated differences in expression of 940 microRNAs (miRNAs), environmentally responsive small non-coding genes that regulate expression of genes at the post-transcriptional level, in ovarian tissue from 8-week-old female C57BL/6 mice exposed to room air or cigarette smoke 5 days per week for 8 weeks. A total of 152 miRNAs were dysregulated in expression, 17 of which were examined with quantitative polymerase chain reaction analysis. Using an online miRNA database tool, complete lists of predicted miRNA gene targets were generated, 12 of which were measured for their expression levels with quantitative polymerase chain reaction. An online bioinformatics resource database, DAVID generated functional classification lists of the target genes and their associated biological pathways. Results of the present pilot study suggest that miR-379, miR-15b, miR-691, miR-872 and miR-1897-5p are potentially useful markers of ovarian toxicity and dysfunction. Examination of the expression pattern of the target mRNA for these miRNA species demonstrated that cigarette smoke exposure induced significant changes that affect mitogen-activated protein kinase signaling pathways. We therefore suggest that miRNAs could serve as sensitive markers of ovarian toxicity and elucidate affected pathways., (© 2018 The Authors. Journal of Applied Toxicology Published by John Wiley & Sons Ltd.)

Recently Bergman et al. (2015) took issue with our comments (Lamb et al., 2014) on the WHO-UNEP(1) report entitled the "State of the Science of Endocrine Disrupting Chemicals - 2012" (WHO 2013a). We find several key differences between their view and ours regarding the selection of studies and presentation of data related to endocrine disrupting chemicals (EDCs) under the WHO-IPCS(2) definition (2002). In this response we address the factors that we think are most important: 1. the difference between hazard and risk; 2. the different approaches for hazard identification (weight of the evidence [WOE] vs. emphasizing positive findings over null results); and 3. the lack of a justification for conceptual or practical differences between EDCs and other groups of agents., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Bisphenol A (BPA) is a widely used industrial chemical in the manufacturing of polycarbonate plastic bottles, food and beverage can linings, thermal receipts, and dental sealants. Animal and human studies suggest that BPA may disrupt normal hormonal function and hence, potentially, have negative effects on the human health. While total BPA is frequently reported, it is recognized that free BPA is the biologically active form and is rarely reported in the literature. The objective of this study was to develop a sensitive and improved method for the measurement of free and total BPA in human urine. Use of a labeled conjugated BPA (bisphenol A-d6 β-D-glucuronide) allowed for the optimization of the enzymatic reaction and permitted an accurate determination of the conjugated BPA concentration in urine samples. In addition, a (13)C12-BPA internal standard was used to account for the analytical recoveries and performance of the isotope dilution method. Solid-phase extraction (SPE) combined with derivatization and analysis using a triple quadrupole GC-EI/MS/MS system achieved very low method detection limit of 0.027 ng/mL. BPA concentrations were measured in urine samples collected during the second and third trimesters of pregnancy in 36 Canadian women. Total maternal BPA concentrations in urine samples ranged from not detected to 9.40 ng/mL (median, 1.21 ng/mL), and free BPA concentrations ranged from not detected to 0.950 ng/mL (median, 0.185 ng/mL). Eighty-six percent of the women had detectable levels of conjugated BPA, whereas only 22 % had detectable levels of free BPA in their urine. BPA levels measured in this study agreed well with data reported internationally.

This study assessed the influence of cigarette smoke condensate (CSC) and benzo(a)pyrene [B(a)P] on the levels of two oxidative stress biomarkers [8-isoprostane (8-IsoP) and 8-hydroxy-2-deoxy Guanosine (8-OH-dG)], in in-vitro spent media of follicle cells. Follicles (100-130 µm) isolated from ovaries of F₁ hybrid (C57Bl/6j × CBA/Ca) mice were cultured for 13 days in media exposed to B(a)P [0 ng ml⁻¹ (control) to 45 ng ml⁻¹] or CSC [0 µg ml⁻¹ (control) to 130 µg ml⁻¹]. The concentrations of oxidative stress biomarkers in spent media were quantified by enzyme-linked immune sorbent assays (ELISA). CSC and B(a)P treatment induced a significant, dose-dependent increase in the concentrations of 8-IsoP and 8-OH-dG in the spent media. We conclude that CSC and B(a)P exposure can induce oxidative stress in ovarian follicles, an effect that may contribute to the previously documented decline in follicle development and premature ovarian failure in women who smoke., (© Her Majesty the Queen in Right of Canada 2013.)

Benzo[a]pyrene (B[a]P) is a prototypical polycyclic aromatic hydrocarbon (PAH) present in cigarette smoke. We previously showed that B[a]P adversely affects follicular development and survival. The objective of this study was to identify the key molecular pathways underlying B[a]P-induced abnormal follicular development. Isolated follicles (100-130 μm) from ovaries of F1 hybrid (C57BL/6j×CBA/Ca) mice were cultured for 8 (preantral/antral follicles) and 12 (preovulatory follicles) days in increasing concentrations of B[a]P (0 ng/mL [control] to 45 ng/mL). Expression of aryl hydrocarbon receptor (AhR), aryl hydroxylase steroidogenic enzyme, cell-cycle, and apoptotic genes were quantified. B[a]P exposure significantly (P<0.05) increased mRNA expression of Cyp1a1 in preantral/antral follicles and Cyp1b1, Bax and Hsp90ab1 in preovulatory follicles. No significant effect on mRNA expression of StAR, Cyp11a1, aromatase, Cdk4, Cdk2, Ccnd2, cIAP2, and survivin was observed. In conclusion, this study suggests that B[a]P exposure significantly affects the phase I enzymes and cell death genes during preantral/antral and preovulatory growth, and thus highlight the AhR signaling and apoptotis pathways in delayed follicle growth and decreased viability., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Anti-NMDA receptor encephalitis is a newly characterized severe neuroautoimmune syndrome with a progressive, clinical course. Most often seen in females, it usually begins with a prodromal phase suggestive of an acute or subclinical upper respiratory tract infection that lasts for up to 2 weeks. This is followed by a psychotic and seizure phase in which the child may rapidly develop seizures, behavioral changes, and, less commonly in children, psychiatric symptoms, resulting in frequent misdiagnoses. The child may become mute and unresponsive but awake during the akinetic phase. Autonomic instability characterizes the hyperkinetic phase. A teratoma or, more rarely, another tumor type is found in 25% of affected adolescents beyond the first decade of life. The finding of oligoclonal protein electrophoresis (>80%) and antibodies in serum and cerebrospinal fluid directed against the NR1 subunit of the NMDA receptor confirms the diagnosis. Prognosis is improved with the appropriate use of immunosuppressant therapies. Relapses in children may be multiple and occur in 20-25% of cases. Recovery is slow and may take 3 years or longer. Even so, the child may not always regain its premorbid level of health., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Bisphenol-A (BPA) has estrogenic properties both in vitro and in vivo. We investigated its impacts upon uterine morphology and estrogen and progesterone receptors after injection on gestational days 1-4 in doses known to disrupt pregnancy. Blastocyst implantation was significantly reduced by doses of 6.75 and 10.125 mg/animal. Uterine luminal area expanded substantially in response to increasing doses of BPA. Luminal epithelial cell height increased following exposure to 10.125 mg/animal, whereas there were no differences in the number of corpora lutea among conditions. The proportion of cells staining positively for estrogen receptors was affected non-monotonically, showing highest levels at 3.375 mg/animal and lowest levels at 10.125 mg/animal. Similarly progesterone receptor expression measured through western blots related non-monotonically to dose, being highest at 3.375 mg/animal and diminishing with increasing dose. These results suggest that BPA exposure during early gestation acts at the uterus to disrupt intrauterine implantation, consistent with an estrogenic effect., ((c) 2010 Elsevier Inc. All rights reserved.)

Background: A single in utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on gestation day 15 decreased epididymal sperm count in adult rats and thus was used to establish a tolerable daily intake for TCDD. However, several laboratories have been unable to replicate these findings. Moreover, conflicting reports of TCDD effects on daily sperm production suggest that spermatogenesis may not be as sensitive to the adverse effects of TCDD as previously thought., Data Sources: We performed a PubMed search using relevant search terms linking dioxin exposure with adverse effects on reproduction and spermatogenesis., Data Synthesis: Developmental exposure to TCDD is consistently linked with decreased cauda epididymal sperm counts in animal studies, although at higher dose levels than those used in some earlier studies. However, the evidence linking in utero TCDD exposure and spermatogenesis is not convincing., Conclusions: Animal studies provide clear evidence of an adverse effect of in utero TCDD exposure on epididymal sperm count but do not support the conclusion that spermatogenesis is adversely affected. The mechanisms underlying decreased epididymal sperm count are unknown; however, we postulate that epididymal function is the key target for the adverse effects of TCDD.

The objectives of this study were to: (1) determine the dosing range necessary to produce serum levels of dieldrin in mice representative of human body burdens; and (2) define the effect of developmental exposure to environmentally relevant concentrations of dieldrin on mammary gland development. Sexually mature female BALB/c mice (n=140) were randomly assigned to receive vehicle, 0.45, 2.25, 4.5, and 22.5 microg dieldrin/g body weight (BW)/day. Serum levels of dieldrin were quantified by gas chromatography in pooled samples (n=4/treatment group). Target levels of 10-30 ng/ml were achieved in 0.45 and 2.25 microg/g dose groups by the end of 2 weeks of treatment. Vehicle or dieldrin (0.45, 2.25, and 4.5 microg/g BW) was administered weekly to sexually mature female BALB/c mice (n=48) throughout mating, pregnancy, and lactation. Treatments had no effect on fertility parameters in dams or mammary gland morphology at sexual maturity. Developmental exposure to dieldrin has no effect on mammary gland development in aged BALB/c mice.

Epidemiological evidence suggests that exposure to the pesticide dieldrin, is associated with increased risk of breast cancer and mortality. We hypothesize that dieldrin promotes breast cancer by increasing survival of breast cancer cells. The aims of this study were to measure the effect of dieldrin on survival of breast cancer cells and the expression of tyrosine kinase B (TrkB), a suppressor of anoikis (apoptosis triggered by inappropriate anchorage). The human breast cancer cell line MDA-MB-231 was treated with dieldrin and proliferation, viability and resistance to anoikis were measured. TrkB expression was measured by Western blot in lysates and by immunohistochemistry in human tissue specimens. Dieldrin increased resistance to anoikis and TrkB expression. TrkB was expressed in a subset of high-grade breast carcinoma specimens. Our results demonstrate that dieldrin increases resistance to anoikis and expression of TrkB and show for the first time TrkB protein expression in human breast cancer.

Endometriosis is an estrogen-dependent disease characterized by the growth of endometrial cells in ectopic locations. Although the etiology of endometriosis is unknown, several hypotheses have been proposed to explain its origin. Retrograde menstruation of endometrial cells into the peritoneum is the most widely accepted theory, however, this phenomenon occurs in approximately 90% of women while the prevalence of endometriosis is much lower. Hence, other factors are thought to contribute to the development of this disease, including exposure to environmental toxicants. Although the epidemiological evidence is equivocal, animal and experimental investigations provide a basis for the proposed association between dioxin and dioxin-like chemical exposure and endometriosis. However, the mechanism(s) underlying this potential association are poorly understood. Development of novel animal models that more reliably recapitulate the pathogenesis and pathophysiology of this disease provide exciting opportunities to further test the link between exposure to these chemicals and endometriosis. Moreover, differential expression of several novel genes that may be important in the disease provides new targets to test the actions of environmental toxicants in the pathobiology of endometriosis.

Background: The adverse effects of cigarette smoking on human fertility have been well documented. However, the mechanism(s) underlying the detrimental effects of cigarette smoking are unknown. Using a novel isolated rat follicle culture assay, we tested the hypothesis that benzo-[a]-pyrene (B[a]P), a constituent of cigarette smoke, can inhibit follicle growth., Methods: B[a]P levels were quantified in the serum and follicular fluid (FF) of women undergoing in vitro fertilization (IVF) treatment exposed to mainstream smoke (n = 19) and non-smokers (n = 10) by gas chromatography mass spectrometry. Isolated rat follicles were cultured with increasing concentrations of B[a]P (1.5-300 ng ml(-1)) and follicle diameter was measured daily., Results: Mean ( +/- Standard error of the mean) B[a]P) was quantified in the serum (0.40 +/- 0.13 ng ml(-1)) and FF (1.32 +/- 0.68 ng ml(-1)) of women who smoke. IVF stimulation and outcome measures were similar between female smokers and non-smokers with the exception of implantation rate and pregnancy rate, which were both significantly lower (P < 0.05) in the MS group. B[a]P treatment significantly reduced rat follicle diameter and attenuated FSH stimulated growth in a dose-dependent manner, beginning at 1.5 ng ml(-1)., Conclusions: Our data suggest that B[a]P, at levels representative of those measured in human FF, may adversely affect follicle development and be an ovarian toxicant.

Tyrosine kinase receptor B (TrkB) gene expression, a neurotrophic factor receptor expressed in the brain and ovary, has recently been identified in deep infiltrating endometriosis by gene array. TrkB is thought to be important in resistance to anchorage independent apoptosis (ANOIKIS) and thus could be important in the pathogenesis of endometriosis. However, TrkB protein expression in the eutopic endometrium of women with and without endometriosis is unknown. Therefore, we examined TrKB protein expression in the endometrium by Western blot (n = 50) and immunohistochemistry (n = 17). Immunoblots of endometrial biopsies were prepared from women with endometriosis (n = 21) vs. healthy controls (n = 29) undergoing benign gynecological surgery at McMaster University Medical Centre. TrkB protein expression was significantly higher in immunoblots from women with endometriosis compared to women without endometriosis. In samples of archived paraffin-embedded endometrial tissue TrkB was localized to the cytoplasm of epithelial cells of the eutopic endometrium from women with endometriosis (n = 7) and without endometriosis (n = 10). We conclude that TrkB protein is expressed in human endometrium. Our results also suggest that TrkB expression may be greater in women with endometriosis compared to women without endometriosis.

Objective: Developmental exposure to dioxin-like compounds has been associated with cognitive and motor impairments in children. These toxicants have been shown to be thyroid toxicants in animal studies. Therefore, the objective of this study was to quantify the overall dioxin-like activity in maternal serum and determine the association between dioxin-like activity and thyroid hormone levels., Study Design: Cross-sectional examination of serum from pregnant women (n = 150) attending a prenatal diagnosis clinic between January 2002 and December 2003., Results: Serum dioxin-like activity was measured in 145 of 150 (96.7%) maternal serum samples. The mean (+/- SEM) serum lipid-adjusted dioxin-like activity was 0.34 +/- 0.01 pg/g. Multiple regression analysis failed to demonstrate a relationship between maternal serum dioxin-like activity and serum thyroid hormone levels., Conclusion: Dioxin-like activity is quantifiable in an overwhelming majority of second-trimester maternal serum samples but there was no relationship between dioxin-like activity and thyroid hormone levels in our study population.

Background: Cigarette smoking (CS) is a widely recognized health hazard, yet it remains prevalent in society and the effects of environmental tobacco smoke exposure on fertility are unknown. Our objective was to measure the effects of CS on the fertility of mainstream (MS) or sidestream (SS) smoke-exposed women compared to their non-smoking (NS) counterparts., Methods: This retrospective study investigated 225 female patients undergoing IVF (n = 97) or ICSI (n = 128). Patients were grouped based on their smoking status for comparison. This included: 39 MS (18 IVF and 21 ICSI); 40 SS (16 IVF and 24 ICSI); and 146 NS (63 IVF and 83 ICSI) women. Fertility treatment outcomes including embryo quality, implantation and pregnancy rate were measured., Results: No difference in embryo quality between the three groups was observed. However, there was a significant difference in implantation rate (MS = 12.0%, SS = 12.6%, and NS = 25.0%) and pregnancy rate (MS = 19.4%, SS = 20.0%, and NS = 48.3%) per embryo transfer., Conclusions: Despite similar embryo quality there was a striking difference in implantation and pregnancy rates of MS and SS smokers when compared with NS. Our data demonstrate that the effects of SS smoking are equally as damaging as MS smoke on fertility.

Environmental toxicants are thought to play a role in several estrogen-dependent diseases including breast cancer and endometriosis. Toxicant-induced increased aromatase activity, an enzyme complex that catalyzes the final rate-limiting step in the conversion of androgens to estrogens, has been reported in assays using placental microsomes and cancer cells in vitro. These data suggest that environmental toxicants can increase aromatase activity and thus increase local tissue estrogen levels, which could have implications for estrogen- dependent functions in target tissues. The objective of this study was therefore to quantify the effect of the stable breakdown product of DDT, 2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE), a toxicant broadly detected in human adipose tissue, serum and follicular fluid, on aromatase activity in the endometrium, an estrogen-sensitive target tissue. Specifically, the effect of increasing log concentrations of p,p'-DDE on aromatase activity was determined in cultures of endometrial stromal cells (ESC). Relative to controls p,p'-DDE treatment significantly increased aromatase activity in ESC (135%). Moreover, ESC cells treated with p,p'-DDE were immunopositive for aromatase, whereas no aromatase staining could be demonstrated in control cultures. Our data demonstrate that p,p'-DDE treatment can increase aromatase activity in ESC in culture.

Reproductive function has been shown to be sensitive to changes in the physical, psychosocial and chemical environments. Although reproductive effects of occupational exposure to hazardous chemicals have been well documented in the literature, the potential effects of chemical contaminants at levels representative of contemporary exposures in the general population are much less certain. Evidence for adverse effects of exposure to environmental contaminants is more conclusive among the lower animals than for humans where considerable controversy remains. In addition to potential reproductive hazards of exposure to environmental contaminants, there is also evidence for adverse reproductive effects of the physical and psychosocial environments. In this review we focus on the difficulties involved in linking exposure to putative hazardous substances in environmental and occupational settings to adverse reproductive outcomes, especially success of IVF procedures. We highlight the plausibility of adverse events through animal and cell studies and the application of these results to the interpretation of human data. We consider both the male and female partners since it is essentially their combined contributions of gametes which may be affected by chemicals, which lead to successful outcomes.

Water chlorination results in the formation of trihalomethanes (THMs) including chloroform. In human stud-ies, fetal growth restriction has been associated with exposure to THMs during pregnancy and impaired fetal growth has been associated with an increased risk of type 2 diabetes. Therefore, the objective of this study was to determine the effect of in utero and lactational exposure to chloroform on birthweight and postnatal indicators of type 2 diabetes. Female Wistar rats were given chloroform (0 microg/L, 75 microg/L) in their drinking water for 2 wk prior to mating until parturition (in utero exposure only) or until weaning (in utero+lactational exposure). At postnatal d 1 (PND1) pups of dams exposed to chloroform had significantly higher serum glucose levels and lower insulin levels, but this effect was not due to b-cell depletion in the neonatal pancreas. Glucose homeostasis in response to a glucose challenge was not changed by chloroform treatment. Chloroform exposure did not affect birthweight; however, offspring of dams exposed to chloroform had significantly impaired postnatal growth. Although fetal and neonatal exposure to chloroform did not elicit physiological changes associated with the onset of type 2 diabetes, there were physiological changes resulting in impaired postnatal growth.