Circulating CD200 is increased in the secretory phase of women with endometriosis as is endometrial mRNA, and endometrial stromal cell CD200R1 is increased in spite of reduced mRNA.

PROBLEM: Estrogen-dependent extrauterine implantation and growth of menstrual endometrial tissue affects roughly 10% of reproductive age women and depends on suppression of local innate immune defenses to prevent ectopic tissue rejection. Immunohistochemistry has shown the immune check-point inhibitor CD200 which can suppress rejection is expressed in eutopic endometrium and in ectopic deposits. Soluble CD200 accumulated in venules draining eutopic and ectopic endometrium of endometriosis cases in the secretory phase but not proliferative phase of the menstrual cycle, and should be increased in the circulation. METHOD OF STUDY: Sera from endometriosis and non-endometriosis controls were tested by ELISA for CD200. Endometrial CD200, CD200R1 and CD200R2 mRNA in eutopic was quantified by RT-PCR and localized by in situ hybridization. CD200R1 protein was quantified by immunohistochemistry. RESULTS: Secretory phase serum CD200 was elevated in women with endometriosis compared to controls. Serum CD200 correlated with matched endometrial CD200 mRNA levels. Expression of mRNA for CD200R1 which signals immune suppression was decreased whereas mRNA for the CD200R2 activating receptor was increased. In situ staining of CD200R1 and CD200R2 mRNA showed both receptors were expressed and the fraction of CD200R that is CD200R1 was reduced in secretory and menstrual phase endometriosis endometrium consistent with the RT-PCR result. By contrast, CD200R1 protein and CD200R1 fraction of total CD200R protein were increased in endometriosis. CONCLUSIONS: Failure to suppress circulating CD200 levels in the secretory phase had an 87% specificity and 90% sensitivity for endometriosis. CD200 and increased CD200R1 expression may facilitate development of ectopic deposits by suppressing rejection mechanisms.

CD200S-positive granulated lymphoid cells in endometrium appear to be CD56-positive uterine NK cells.

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.

Endometrial microbiota is more diverse in people with endometriosis than symptomatic controls.

Endometriosis is a chronic, estrogen-dependent gynecological condition affecting approximately 10% of reproductive age women. The most widely accepted theory of its etiology includes retrograde menstruation. Recent reports suggest the uterus is not sterile. Thus, the refluxed menstrual effluent may carry bacteria, and contribute to inflammation, the establishment and growth of endometriotic lesions. Here, we compared and contrasted uterine bacteria (endometrial microbiota) in people with surgically confirmed presence (N = 12) or absence of endometriosis (N = 9) using next-generation 16S rRNA gene sequencing. We obtained an average of > 9000 sequence reads per endometrial biopsy, and found the endometrial microbiota of people with endometriosis was more diverse (greater Shannon Diversity Index and proportion of 'Other' taxa) than symptomatic controls (with pelvic pain, surgically confirmed absence of endometriosis; diagnosed with other benign gynecological conditions). The relative abundance of bacterial taxa enriched in the endometrial microbiota of people with endometriosis belonged to the Actinobacteria phylum (Gram-positive), Oxalobacteraceae (Gram-negative) and Streptococcaceae (Gram-positive) families, and Tepidimonas (Gram-negative) genus, while those enriched in the symptomatic controls belonged to the Burkholderiaceae (Gram-negative) family, and Ralstonia (Gram-negative) genus. Taken together, results suggest the endometrial microbiota is perturbed in people with endometriosis.

Factors affecting stability of plasma brain-derived neurotrophic factor.

Circulating concentrations of brain-derived neurotrophic factor (BDNF) have been linked to cancer, neuropsychiatric, diabetes, and gynecological disorders. However, factors influencing plasma storage and subsequent BDNF quantification are incompletely understood. Therefore, the anticoagulant used in plasma separator tubes, storage-time, storage-temperature, and repeated freeze-thaw cycles on circulating BDNF concentrations was evaluated. Peripheral blood samples were collected from healthy women (n = 14) and men (n = 10) recruited prospectively from McMaster University (August 2014). Blood was collected from the cubital vein into plasma separator tubes containing five different anticoagulant systems [K2EDTA, Li-Hep, Li-Hep (gel), Na-Hep, Na-Hep (glass)], and placed on ice for transport to the lab for centrifugation. Plasma samples (n = 16) collected in K2EDTA tubes from women recruited to a previous study (April 2011 to December 2012) were used to determine the effect of multiple freeze-thaw cycles. Plasma BDNF was quantified using a commercially available ELISA kit. Plasma concentrations of BDNF were significantly affected by the type of plasma separator tube, storage-time, and number of freeze-thaw cycles. Storage temperature (- 20 vs. - 80 °C) did not significantly affect the quantity of BDNF measured as mean BDNF concentrations generally fell within our calculated acceptable change limit up to 6 months in the freezer. Our results suggest that for quantification of circulating BDNF blood collected in K2EDTA tubes and plasma stored up to 6 months at either - 20 or - 80 °C produces reproducible results that fall within an acceptable range. However, plasma samples stored beyond 6 months and repeated freeze-thaw cycles should be avoided.

A Selective and Sensitive Gas Chromatography-Tandem Mass Spectrometry Method for Quantitation of Synthetic Musks in Human Serum.

BACKGROUND: Synthetic musk compounds are widely used as fragrances in many consumer products; however, information on human exposure and health effects is limited. Also, analytical methods for their quantification in biological matrices are limited. OBJECTIVE: In this study, an integrated method was developed and validated for the analysis of selected synthetic musk compounds in human serum. METHOD: The method is based on liquid-liquid extraction (LLE), sample clean-up by solid-phase extraction (SPE), and separation and detection by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). RESULTS: The method demonstrated good recoveries (86-105%) and high sensitivity, with low method detection limits (MDLs) ranging from 0.04 to 0.17 µg/L. The method was applied to the analysis of 10 synthetic musk compounds in 40 serum samples collected from Canadian women aged 20-44 years (20 individual samples collected in 2014 and 20 pooled samples collected in 2006). The most commonly detected compound was Galaxolide (HHCB), with median concentrations of 0.59 µg/L in samples collected in 2006, and 0.34 µg/L for samples collected in 2014. Musk ketone (MK) was not detected in any of the samples collected in 2006, but was detected in 60% of the samples collected in 2014 with a median concentration of 0.29 µg/L. Tonalide (AHTN) was detected in only one sample above its MDL (0.12 µg/L). CONCLUSIONS: This is the first study in Canada to report levels of synthetic musks in human. The data generated from this study has been used in risk screening assessment by Environment and Climate Change Canada and Health Canada.

Identification of candidate microRNA markers of endometriosis with the use of next-generation sequencing and quantitative real-time polymerase chain reaction.

OBJECTIVE: To identify novel candidate diagnostic microRNA (miRNA) markers of endometriosis by means of an unbiased search with confirmation by means of targeted polymerase chain reaction (PCR). DESIGN: Retrospective cohort. SETTING: University teaching hospitals. PATIENT(S): Women with endometriosis and control women, confirmed with the use of laparoscopy. INTERVENTIONS(S): Diagnostic laparoscopy and blood sample. MAIN OUTCOME MEASURE(S): Next-generation sequencing (NGS) and quantitative real-time PCR (qRT-PCR). RESULT(S): Candidate miRNAs differentially expressed in women with endometriosis compared with control women were identified by means of NGS and selected for qRT-PCR. Plasma samples from another cohort of women with surgically confirmed endometriosis (n = 53) and disease-free control women (n = 53) were checked for hemolysis using spectrophotometry and the ratio of miR-23a and miR-451 by means of qRT-PCR. MicroRNA signatures were quantified by means of qRT-PCR in hemolysis-free plasma samples of case subjects (n = 25) and control subjects (n = 28) with the use of miRcury LNA miRNA. Circulating levels of eight miRNAs (miR-199a-3p, miR-143-3p, miR-340-5p, let-7b-5p, miR-21-5p, miR-17-5p, miR-20a-5p, and miR-103a-3p) were significantly lower in case subjects compared to control subjects. The sensitivity and specificity for individual miRNAs ranged from 0.36 to 1.00 and from 0.43 to 1.00, respectively, but when combined produced sensitivity and specificity of 0.92 and 0.86 with positive (PPV) and (NPV) predictive values of 0.85 and 0.92, respectively. However, combination of five miRNAs (miR-17-5p, miR-20a-5p, miR-199a-3p, miR-143-3p, and let-7b-5p) produced sensitivity and specificity of 0.96 and 0.79 with PPV and NPV of 0.80 and 0.96, respectively. CONCLUSION(S): We conclude that a panel of candidate miRNAs was comparable to laparoscopy in distinguishing between women with endometriosis and control women.

Prenatal perfluoroalkyl substances and newborn anogenital distance in a Canadian cohort.

Exposure to the man-made chemicals perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS) and perfluorohexanesulfonate (PFHxS) is widespread. These perfluoroalkyl substances (PFASs) have been associated with androgenic endocrine-disrupting properties; however, the evidence is equivocal and few human studies have examined the association between prenatal exposure to PFASs and markers of androgenic endocrine disruption such as changes in anogenital distance (AGD). In the MIREC cohort, PFOA, PFOS and PFHxS were analyzed in first trimester maternal plasma. AGD was measured in 205 male and 196 female newborns. The change in estimate procedure was used to identify confounders by sex and AGD in multiple linear regression models. Geometric mean plasma concentrations (95% CI) for PFOA, PFOS and PFHxS were 1.71 (1.61, 1.81), 4.40 (4.18, 4.64) and 1.15 (1.06, 1.25) µg/L, respectively. A one-unit increase in natural log transformed PFOA was associated with a 1.36 mm (95% CI 0.30, 2.41) increase in anoscrotal distance, adjusting for household income, active smoking status during pregnancy and gestational age. However, when examined by quartiles, a non-monotonic pattern was observed with wide confidence intervals. No consistent patterns were observed between maternal PFAS concentrations and female AGDs. This study found no clear evidence that maternal plasma concentrations of PFOS, PFOA or PFHxS were associated with shorter infant anogenital distance in males or any change in AGD in females. Whether the positive association observed between longer anoscrotal distance and PFOA is real or would have any long-lasting effect on the reproductive health of males is unknown and needs to be investigated further.

MicroRNA expression pattern differs depending on endometriosis lesion type.

MicroRNA (miRNA), noncoding segments of RNA involved in post-transcriptional regulation of protein expression are differentially expressed in eutopic endometrium of women with and without endometriosis compared to endometriotic lesions. However, endometriotic lesion types are known to be biochemically distinct and therefore hypothesized that miRNAs are differentially expressed in endometriomas compared to peritoneal and deep-infiltrating lesions. Therefore, endometrial biopsies and ectopic implants from women (n = 38) undergoing laparoscopic surgery for chronic pelvic pain were collected. Samples of endometriomas, peritoneal or deep-infiltrating lesions were selected from our tissue bank for study participants who exclusively had only one lesion type noted on their surgical report. Quantitative real-time polymerase chain reaction for miR-9, miR-21, miR-424, miR-10a, miR-10b, and miR-204 was performed. miR-204 expression was significantly lower (P = 0.0016) in the eutopic endometrium of women with endometriosis compared to controls. Relative expression of miR-21, miR-424, and miR-10b differed significantly (P < 0.05) across endometriotic lesion types. Finally, all miRNAs isolated from endometriomas, peritoneal and deep-infiltrating lesions studied were differentially expressed compared to matched eutopic endometrium samples. We therefore conclude that miRNA expression in the eutopic endometrium from women with endometriosis differs from symptomatic controls. Moreover, miRNA expression pattern is dependent on the endometriotic lesion type studied. We suggest that identification of different miRNA expression patterns for endometriomas, peritoneal and deep-infiltrating lesions could contribute to individualized patient care for women with endometriosis.

Identification of microRNAs as potential markers of ovarian toxicity.

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.

Screening for DNA adducts in ovarian follicles exposed to benzo[a]pyrene and cigarette smoke condensate using liquid chromatography-tandem mass spectrometry.

A rapid mass spectrometric method was applied to non-targeted screening of DNA adducts in follicular cells (granulosa cells and theca cells) from isolated ovarian follicles that were exposed in-vitro to benzo[a]pyrene (B[a]P) and cigarette smoke condensate (CSC) for 13days of culture. The method employed a constant neutral loss (CNL) scan to identify chromatographic peaks associated to a neutral loss of deoxyribose moiety of DNA nucleosides. These peaks were subsequently analyzed by a product ion scan in tandem mass spectrometry to elucidate structures of DNA adducts. The identification was further confirmed through synthesis of proposed DNA adducts where possible. Three DNA adducts, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide-dG (BPDE-dG), phenanthrene 1,2-quinone-dG (PheQ-dG) and B[a]P-7,8-quinone-dG (BPQ-dG) were identified in the follicular cells from isolated ovarian follicles exposed to B[a]P. Along with these three, an additional DNA adduct, 4-aminobiphenyl-dG, was identified in the follicular cells from isolated ovarian follicles exposed to CSC. The amounts of the identified DNA adducts in follicular cells increased in a dose-dependent manner for both B[a]P (0, 1.5, 5, 15 and 45ng/mL) and CSC (0, 30, 60, 90 and 130µg/mL). The results revealed that B[a]P-related DNA adducts were the major adducts in the ovarian follicular cells exposed to CSC. The results also revealed that two oxidative biomarkers, 8-hydroxy-2-deoxy guanosine (8-OH-dG) and 8-isoprostane (8-IsoP), in both B[a]P-exposed and CSC-exposed ovarian follicles had strong correlations with the three DNA adducts, BPDE-dG, BPQ-dG and PheQ-dG. A pathway to describe formation of DNA adducts was proposed based on the DNA adducts observed.

Effects of in vitro exposure to butylparaben and di-(2 ethylhexyl) phthalate, alone or in combination, on ovarian function.

Parabens and phthalates are commercial chemicals widely used in the manufacture of industrial and consumer products frequently found as contaminants in biological fluids. We evaluated the effects of di-(2-ethylhexyl) phthalate (DEHP) (ranging from 10(-9) to 10(-7) m [1-100 nm; 0.39-39 ng ml(-1) ]) and butylparaben (BP) (ranging from 10(-8) to 10(-5) m [10 nm-10 µm; 1.9 ng ml(-1) to 1.9 µg ml(-1) ]), alone and in combination, on isolated mouse preantral follicle and human granulosa cell (hGC) cultures to study direct effects on follicle growth and ovarian steroidogenesis. Our results revealed that, in follicle culture, DEHP and BP attenuate estradiol output but only when present together. DEHP decreases progesterone concentrations in the spent media of hGC cultures, an effect that was attenuated when BP was added together with DEHP. Although changes in steroidogenesis were observed, no effects on follicular development or survival were noted in the culture systems. We suggest that BP and DEHP act with additive effect to decrease estradiol production whereas at later stages of follicle development BP blocks the effect of DEHP in hGCs resulting in decreased progesterone output. Taken together our results suggest that DEHP and BP adversely affect steroidogenesis from the preantral stage onward and the effects of these chemicals are both stage-dependent and modified by co-exposure. Copyright © 2016 John Wiley & Sons, Ltd.

Concentrations of persistent organic pollutants in maternal and cord blood from the maternal-infant research on environmental chemicals (MIREC) cohort study.

BACKGROUND: Pregnant women are an especially important population to monitor for environmental exposures given the vulnerability of the developing fetus. During pregnancy and lactation chemical body burdens may change due to the significant physiological changes that occur. Developmental exposures to some persistent organic pollutants (POPs) have been linked with adverse health outcomes. METHODS: First trimester maternal and cord blood plasma concentrations of several POPs including polychlorinated biphenyls (PCBs), organochlorine pesticides (OCs), polybrominated diphenyl ethers (PBDE)s and perfluoroalkyl substances (PFASs) were measured in samples from 1983 pregnant women enrolled in the Maternal-Infant Research on Environmental Chemicals (MIREC) cohort. Predictors of exposure were also identified. RESULTS: In maternal plasma, there was >90 % detection for the perfluoroalkyl substances (PFASs) perfluorooctanoic acid (PFOA), perfluoroctane sulfonate (PFOS), perfluorohexane sulfonate (PFHxS), and dichlorodiphenyldichloroethylene (DDE), oxychlordane and PCB 138 and 153. Cord blood plasma had much lower detection rates with low or very limited detection for most PCBs and PBDEs. The PFASs were the most frequently detected (23-64 %) chemical class in cord plasma. In a subset of 1st and 3rd trimester paired samples, PFAS concentrations were found to be strongly correlated and had ICCs ranging from 0.64 (PFOA) to 0.83 (PFHxS). The cord:maternal plasma concentration ratios ranged from 0.14 (PFOS) to 0.87 (oxychlordane, lipid adjusted). Similar to other studies, we found parity, maternal age, income, education, smoking status, pre-pregnancy BMI and fish consumption to be significant predictors for most chemicals. Those participants who were foreign-born had significantly higher concentrations of organochlorinated pesticides and PCBs. CONCLUSIONS: In the MIREC study, multiple chemical contaminants were quantified in the plasma of pregnant women. In cord plasma PFOA had the highest detection rate. However, compared to other Canadian and international population studies, the MIREC participants had lower contaminant concentrations of these substances.

Assessing brain-derived neurotrophic factor as a novel clinical marker of endometriosis.

OBJECTIVE: To evaluate novel clinical markers of endometriosis including the neurotrophins brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin 4/5 (NT4/5) and compare them to others previously reported in the literature including cancer antigen 125 (CA-125) and C-reactive protein (CRP). DESIGN: Prospective study. SETTING: University hospital. PATIENT(S): One hundred thirty-eight women were prospectively and consecutively recruited (April 2011-April 2015; cases: undergoing endometriosis surgery, n = 96; controls: benign gynecological surgery, n = 24 combined with healthy women, no history of pelvic pain, not undergoing surgery, n = 18). INTERVENTION(S): Collection of peripheral blood, gynecological and demographic information, eutopic biopsy in women undergoing laparoscopy. MAIN OUTCOME MEASURE(S): Circulating BDNF, NGF, NT4/5, CA-125, and CRP were quantified by ELISA. RESULT(S): Plasma concentrations of BDNF were significantly greater in women with endometriosis (1,091.9 pg/mL [640.4-1,683.1]; n = 68, untreated) than in controls (731.4 pg/mL [352.1-1,176.2]; n = 36), whereas circulating NGF, NT4/5, CA-125, and CRP were not different. When assessed for their ability to differentiate between women with revised Classification of the American Society of Reproductive Medicine stage 1 and 2 or stage 3 and 4 disease and controls, BDNF was the only putative marker able to identify stage 1 and 2 disease, with a sensitivity and specificity of 91.7% and 69.4%, respectively, using an arbitrary cutoff value of 1,000 pg/mL. We also demonstrated that circulating BDNF in women with endometriosis who were receiving ovarian suppression for disease was equivalent to that in the control group. This suggests that BDNF may also offer the opportunity to monitor patient response to treatment. CONCLUSION(S): Plasma BDNF is a potentially useful clinical marker of endometriosis that is superior to NGF, NT4/5, CA-125, and CRP.

Cigarette smoke exposure triggers the autophagic cascade via activation of the AMPK pathway in mice.

We previously demonstrated that cigarette smoke (CS) exposure decreases primordial follicle counts and induces autophagy in ovarian granulosa cells in preference to apoptosis. Therefore, the objective of this study was to investigate molecular targets underlying smoke-induced activation of the reparative autophagy pathway in the ovary. Briefly, ovarian homogenates were prepared from adult female mice exposed to mainstream CS twice daily for 8 wk, using a whole-body exposure system. A gene array revealed that CS exposure induced a greater than 2-fold significant increase in the expression of proautophagic genes Cdkn1b, Map1lc3a, Bad, and Sqstm1/p62. A significant increase in Prkaa2, Pik3c3, and Maplc31b expression, as well as a significant decrease in Akt1 and Mtor expression, was detected by quantitative PCR. The 5'-AMP-activated protein kinase catalytic subunit (AMPK) alpha1 + alpha2 and ATG7 protein expression was significantly increased, whereas AKT1, mTOR, CDKN1B/p27, and CXCR4 proteins were significantly decreased in CS exposed versus control ovaries. Up-regulation of AMPK alpha1 + alpha2, a known initiator of autophagic signaling, and ATG7 further suggests activation of the autophagy cascade. Two prosurvival factors, AKT and mTOR, were decreased in expression, an outcome that favors induction of the autophagy pathway, whereas decreased levels of CDKN1B is suggestive of cell cycle dysregulation. In summary, our data suggest that CS exposure induces ovarian follicle loss through induction of the autophagic cascade via the AMPK pathway together with inhibition of antiautophagic markers AKT and mTOR. We further postulate that toxicant-induced dysregulation of reparative autophagy is a novel pathway central to impaired follicle development and subfertility.

Estrogen induced changes in uterine brain-derived neurotrophic factor and its receptors.

STUDY QUESTION: Are brain-derived neurotrophic factor (BDNF) and its receptors, NTRK2, NGFR and SORT1, 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 AND THE 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 SORT1 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 ≤ 0.001, 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.

Treatment of Endometriosis with the GnRHa Deslorelin and Add-Back Estradiol and Supplementary Testosterone.

BACKGROUND: This randomized, multicenter, open-label clinical trial was intended to generate pilot data on the efficacy and safety of the gonadotropin-releasing hormone agonist (GnRHa) deslorelin (D) with low-dose estradiol ± testosterone (E2 ± T) add-back for endometriosis-related pelvic pain. METHODS: Women with pelvic pain and laparoscopically confirmed endometriosis were treated with a six-month course of daily intranasal D with concurrent administration of either transdermal E2, intranasal E2, or intranasal E2 + T. Efficacy data included evaluation of dyspareunia, dysmenorrhea, pelvic pain, tenderness, and induration. Cognition and quality of life were also assessed. Safety parameters included assessment of endometrial hyperplasia, bone mineral density (BMD), and hot flashes. RESULTS: Endometriosis symptoms and signs scores decreased in all treatment arms from a baseline average of 7.4 to 2.5 after 3 months of treatment and 3.4 after 6 months. BMD changes and incidence of hot flashes were minimal, and no endometrial hyperplasia was observed. Patient-reported outcomes showed significant improvement across multiple domains. CONCLUSIONS: Daily intranasal D with low dose E2 ± T add-back resulted in significant reduction in severity of endometriosis symptoms and signs with few safety signals and minimal hypoestrogenic symptoms that would be expected with the use of a GnRHa alone.

Phthalate and bisphenol A exposure among pregnant women in Canada--results from the MIREC study.

Bisphenol A (BPA) and phthalates are endocrine disruptors possibly linked to adverse reproductive and neurodevelopmental outcomes. These chemicals have commonly been measured in urine in population surveys; however, such data are limited for large populations of pregnant women, especially for the critical first trimester of pregnancy. The aim of the study was to measure BPA and phthalate metabolites in first trimester urine samples collected in a large national-scale pregnancy cohort study and to identify major predictors of exposure. Approximately 2000 women were recruited in the first trimester of pregnancy from ten sites across Canada. A questionnaire was administered to obtain demographic and socio-economic data on participants and a spot urine sample was collected and analyzed for total BPA (GC-MS/MS) and 11 phthalate metabolites (LC-MS/MS). The geometric mean (GM) maternal urinary concentration of total BPA, uncorrected for specific gravity, was 0.80 (95% CI 0.76-0.85) µg/L. Almost 88% of the women had detectable urinary concentrations of BPA. An analysis of urinary concentrations of BPA by maternal characteristics with specific gravity as a covariate in the linear model showed that the geometric mean concentrations: (1) decreased with increasing maternal age, (2) were higher in current smokers or women who quit during pregnancy compared to never smokers, and (3) tended to be higher in women who provided a fasting urine sample and who were born in Canada, and had lower incomes and education. Several of the phthalate metabolites analyzed were not prevalent in this population (MCHP, MMP, MiNP, MOP), with percentages detectable at less than 15%. The phthalate metabolites with the highest measured concentrations were MEP (GM: 32.02 µg/L) and MnBP (GM: 11.59 µg/L). MBzP urinary concentrations decreased with maternal age but did not differ by time of urine collection; whereas the DEHP metabolites tended to be higher in older women and when the urine was collected later in the day. This study provides the first biomonitoring results for the largest population of pregnant women sampled in the first trimester of pregnancy. The results indicate that exposure among this population of pregnant women to these chemicals is comparable to or even lower than that observed in a Canadian national population-based survey.

The brain-uterus connection: brain derived neurotrophic factor (BDNF) and its receptor (Ntrk2) are conserved in the mammalian uterus.

The neurotrophins are neuropeptides that are potent regulators of neurite growth and survival. Although mainly studied in the brain and nervous system, recent reports have shown that neurotrophins are expressed in multiple target tissues and cell types throughout the body. Additionally, dysregulation of neurotrophins has been linked to several disease conditions including Alzheimer's, Parkinson's, Huntington's, psychiatric disorders, and cancer. Brain derived neurotrophic factor (BDNF) is a member of the neurotrophin family that elicits its actions through the neurotrophic tyrosine receptor kinase type 2 (Ntrk2). Together BDNF and Ntrk2 are capable of activating the adhesion, angiogenesis, apoptosis, and proliferation pathways. These pathways are prominently involved in reproductive physiology, yet a cross-species examination of BDNF and Ntrk2 expression in the mammalian uterus is lacking. Herein we demonstrated the conserved nature of BDNF and Ntrk2 across several mammalian species by mRNA and protein sequence alignment, isolated BDNF and Ntrk2 transcripts in the uterus by Real-Time PCR, localized both proteins to the glandular and luminal epithelium, vascular smooth muscle, and myometrium of the uterus, determined that the major isoforms expressed in the human endometrium were pro-BDNF, and truncated Ntrk2, and finally demonstrated antibody specificity. Our findings suggest that BDNF and Ntrk2 are transcribed, translated, and conserved across mammalian species including human, mouse, rat, pig, horse, and the bat.

In vitro exposure to cigarette smoke induces oxidative stress in follicular cells of F1 hybrid mice.

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.

Novel male exposure reduces uterine e-cadherin, increases uterine luminal area, and diminishes progesterone levels while disrupting blastocyst implantation in inseminated mice.

Exposure to novel male mice disrupts blastocyst implantation in inseminated female mice, and evidence increasingly implicates the female's absorption of male urinary estrogens. We observed implantation sites in male-exposed and isolated control female mice during gestation days (GD) 2-8, observing a significant reduction in male-exposed females compared to controls, particularly on GD 6 and 8. We also measured transitions in uterine luminal area and e-cadherin expression, as these processes are modulated by estrogens. Luminal area was greater in male-exposed females than in controls during the post-implantation period (GD 5-7). E-cadherin levels were suppressed by male exposure, particularly during GD 4-6 Serum progesterone levels were also reduced in male-exposed females. The effects of male exposure on uterine closure and e-cadherin levels are consistent with established effects of estrogens, and suggest a possible mechanism that could contribute to implantation failure. This article is part of a Special Issue entitled 'Pregnancy and Steroids'.