An automated computational data pipeline to rapidly acquire, score, and rank toxicological data for ecological hazard assessment.

Biological Evaluations support Endangered Species Act (ESA) consultation with the US Fish and Wildlife Service and National Marine Fisheries Service by federal action agencies, such as the USEPA, regarding impacts of federal activities on threatened or endangered species. However, they are often time-consuming and challenging to conduct. The identification of pollutant benchmarks or guidance to protect taxa for states and tribes when USEPA has not yet developed criteria recommendations is also of importance to ensure a streamlined approach to Clean Water Act program implementation. Due to substantial workloads, tight regulatory timelines, and the often-protracted length of ESA consultations, there is a need to streamline the development of biological evaluation toxicity assessments for determining the impact of chemical pollutants on ESA-listed species. Moreover, there is limited availability of species-specific toxicity data for many contaminants, further complicating the consultation process. New approach methodologies are being increasingly used in toxicology and chemical safety assessment to rapidly and cost-effectively provide data that can fill gaps in hazard and/or exposure characterization. Here, we present the development of an automated computational pipeline-RASRTox (Rapidly Acquire, Score, and Rank Toxicological data)-to rapidly extract and categorize ecological toxicity benchmark values from curated data sources (ECOTOX, ToxCast) and well-established quantitative structure-activity relationships (TEST, ECOSAR). As a proof of concept, points-of-departure (PODs) generated in RASRTox for 13 chemicals were compared against benchmark values derived using traditional methods-toxicity reference values (TRVs) and water quality criteria (WQC). The RASRTox PODs were generally within an order of magnitude of corresponding TRVs, though less concordant compared with WQC. The greatest utility of RASRTox, however, lies in its ability to quickly and systematically identify critical studies that may serve as a basis for screening value derivation by toxicologists as part of an ecological hazard assessment. As such, the strategy described in this case study can potentially be adapted for other risk assessment contexts and stakeholder needs. Integr Environ Assess Manag 2024;00:1-15. © 2024 Society of Environmental Toxicology & Chemistry (SETAC). This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Evaluating potential developmental toxicity of perfluoroalkyl and polyfluoroalkyl substances in Xenopus laevis embryos and larvae.

As part of the US Environmental Protection Agency's perfluoroalkyl and polyfluoroalkyl substances (PFAS) Action Plan, the agency is committed to increasing our understanding of the potential ecological effects of PFAS. The objective of these studies was to examine the developmental toxicity of PFAS using the laboratory model amphibian species Xenopus laevis. We had two primary aims: (1) to understand the developmental toxicity of a structurally diverse set of PFAS compounds in developing embryos and (2) to characterize the potential impacts of perfluorooctanesulfonic acid (PFOS), perfluorohexanesulfonic acid (PFHxS), perfluorooctanoic acid (PFOA), and hexafluoropropylene oxide-dimer acid (HFPO-DA a.k.a. GenX), on growth and thyroid hormone-controlled metamorphosis. We employed a combination of static renewal and flow-through exposure designs. Embryos were exposed to 17 structurally diverse PFAS starting at the midblastula stage through the completion of organogenesis (96 h). To investigate impacts on PFOS, PFOA, PFHxS, and HFPO-DA on development and metamorphosis, larvae were exposed from premetamorphosis (Nieuwkoop Faber stage 51 or 54) through pro metamorphosis. Of the PFAS tested in embryos, only 1H,1H,10H,10H-perfluorodecane-1,10-diol (FC10-diol) and perfluorohexanesulfonamide (FHxSA) exposure resulted in clear concentration-dependent developmental toxicity. For both of these PFAS, a significant increase in mortality was observed at 2.5 and 5 mg/L. For FC10-diol, 100% of the surviving embryos were malformed at 1.25 and 2.5 mg/L, while for FHxSA, a significant increase in malformations (100%) was observed at 2.5 and 5 mg/L. Developmental stage achieved was the most sensitive endpoint with significant effects observed at 1.25 and 0.625 mg/L for FC10-diol and FHxSA, respectively. In larval studies, we observed impacts on growth following exposure to PFHxS and PFOS at concentrations of 100 and 2.5 mg/L, respectively, while no impacts were observed in larvae when exposed to PFOA and HFPO-DA at concentration of 100 mg/L. Further, we did not observe impacts on thyroid endpoints in exposed larvae. These experiments have broadened our understanding of the impact of PFAS on anuran development.

Endometrial thickness is a good predictor of clinical pregnancy and live birth rates in fresh and frozen-thawed embryo transfer cycles.

OBJECTIVE: The aim of this study was to investigate the effect of the endometrial thickness (EMT) measured on embryo transfer day on clinical pregnancy (CPR), live birth (LBR), and miscarriage rates (MR) in fresh and frozen-thawed embryo transfer cycles. PATIENTS AND METHODS: This prospective cohort study consisted of 160 patients, 80 frozen-thawed and 80 fresh cycles. Endometrial thickness was measured on the day of embryo transfer for fresh and frozen cycles. In addition to the endometrial thickness, the endometrial appearances of the patients in both groups were also recorded. Those without trilaminar appearance were excluded from the study. Both groups were classified according to the EMT values measured on the day of the transfer. The number of groups was calculated considering 1 mm intervals of EMT, and a total of 8 groups were formed. The initial group started with <6 mm, while the final group was >12 mm. The relationship between endometrial thickness, clinical pregnancy, live birth and miscarriage rates was analyzed using multivariable regression analysis. RESULTS: A significant correlation was observed between endometrial thickness values, clinical pregnancy rates, live birth rates in the analyses performed after adjusting for age, infertility duration, body mass index, number of MII oocytes, number and quality of embryos transferred. Based on univariate analysis, each 1 mm increase in EMT resulted in a significant increase in CPR (OR=1.08, 95% CI: 1.07-1.09, p<0.01). Similarly, the increase in EMT led to a significant increase in LBR (OR=1.12, 95% CI: 1.10-1.14, p<0.01). Although the relationship between miscarriage rates and EMT is not as clear as LBR and CPR, the increase in EMT led to a significant reduction in MR (OR=1.05, 95% CI: 1.03-1.05, p=0.03). The lowest CPR was detected at EMT <6 mm, while the EMT value with the highest CPR was 11-12 mm in both groups. Likewise, in both groups, the lowest LBR was detected at EMT <6 mm, while the EMT value with the highest LBR was 11-12 mm. Although MR showed a fluctuating course according to EMT values, it reached its highest rate at EMT <6 mm (100%). In EMT 11-12 mm, MR reached its lowest level (12.5%). If EMT >12 mm, an increase in MR rates was observed again (33.3%). CONCLUSIONS: Clinical pregnancy and live birth rates remain optimal if the endometrial thickness is between 11-12 mm in both fresh and frozen-thawed cycles. A fluctuating course is observed between EMT values and miscarriage rates.

Comparison of in silico, in vitro, and in vivo toxicity benchmarks suggests a role for ToxCast data in ecological hazard assessment.

Large repositories of in vitro bioactivity data such as US EPA's Toxicity Forecaster (ToxCast) provide a wealth of publicly accessible toxicity information for thousands of chemicals. These data can be used to calculate point-of-departure (POD) estimates via concentration-response modeling that may serve as lower bound, protective estimates of in vivo effects. However, the data are predominantly based on mammalian models and discussions to date about their utility have largely focused on potential integration into human hazard assessment, rather than application to ecological risk assessment. The goal of the present study was to compare PODs based on (1) quantitative structure-activity relationships (QSARs), (2) the 5th centile of the activity concentration at cutoff (ACC), and (3) lower-bound cytotoxic burst (LCB) from ToxCast, with the distribution of in vivo PODs compiled in the Ecotoxicology Knowledgebase (ECOTOX). While overall correlation between ToxCast ACC5 and ECOTOX PODs for 649 chemicals was weak, there were significant associations among PODs based on LCB and ECOTOX, LCB and QSARs, and ECOTOX and QSARs. Certain classes of compounds showed moderate correlation across datasets (eg, antimicrobials/disinfectants), while others, such as organophosphate insecticides, did not. Unsurprisingly, more precise classifications of the data based on ECOTOX effect and endpoint type (eg, apical vs biochemical; acute vs chronic) had a significant effect on overall relationships. Results of this research help to define appropriate roles for data from new approach methodologies in chemical prioritization and screening of ecological hazards.

A biologically based computational model for the hypothalamic-pituitary-thyroid (HPT) axis in Xenopus laevis larvae.

A biologically based computational model was developed to describe the hypothalamic-pituitary-thyroid (HPT) axis in developing Xenopus laevis larvae. The goal of this effort was to develop a tool that can be used to better understand mechanisms of thyroid hormone-mediated metamorphosis in X. laevis and predict organismal outcomes when those mechanisms are perturbed by chemical toxicants. In this report, we describe efforts to simulate the normal biology of control organisms. The structure of the model borrows from established models of HPT axis function in mammals. Additional features specific to X. laevis account for the effects of organism growth, growth of the thyroid gland, and developmental changes in regulation of thyroid stimulating hormone (TSH) by circulating thyroid hormones (THs). Calibration was achieved by simulating observed changes in stored and circulating levels of THs during a critical developmental window (Nieuwkoop and Faber stages 54-57) that encompasses widely used in vivo chemical testing protocols. The resulting model predicts that multiple homeostatic processes, operating in concert, can act to preserve circulating levels of THs despite profound impairments in TH synthesis. Represented in the model are several biochemical processes for which there are high-throughput in vitro chemical screening assays. By linking the HPT axis model to a toxicokinetic model of chemical uptake and distribution, it may be possible to use this in vitro effects information to predict chemical effects in X. laevis larvae resulting from defined chemical exposures.

Determination of maternal serum pro-inflammatory cytokine changes in intrauterine growth restriction.

OBJECTIVE: To evaluate maternal serum inflammatory marker changes in intrauterine growth restriction (IUGR) pregnancies. PATIENTS AND METHODS: 50 healthy pregnant women and 50 patients diagnosed with IUGR were enrolled. Maternal serum high sensitivity C-reactive protein (hsCRP), erythrocyte sedimentation rate (ESR), tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-10 levels were measured before delivery and neonatal outcomes were evaluated. RESULTS: Birth weight, Apgar scores, and cord blood gas pH were lower in the IUGR group (p<0.001, p<0.001, p<0.001 and p=0.006, respectively). While the levels of ESR, hsCRP, IL-6, and TNF-α were higher, the IL-10 level was found to be lower in the IUGR group (p<0.001, p=0.033, p<0.001, p=0.004 and p<0.001, respectively). As ESR, hsCRP, and IL-6 levels increased, birth weight, Apgar scores, and cord blood gas pH decreased (p<0.001, p<0.001, p<0.001, p<0.001, p=0.02, p=0.002, p=0.001, p=0.03, p<0.001, p<0.001, p<0.001 and p=0.02, respectively). As TNF-α level increased, only birth weight and Apgar score at the 1st minute decreased (p=0.006 and p=0.048, respectively). As IL-10 level decreased, birth weight, Apgar scores, and cord blood gas pH decreased (p<0.001 for all). IL-6 (>3.2 pg/ml) had a sensitivity of 100%, specificity of 100%, PPV of 100% and NPV of 100%. CONCLUSIONS: While birth weight, Apgar score and cord blood pH decreased in IUGR cases, ESR, hsCRP, IL-6 and TNF-α levels increased. Combined measurement of these markers can be used for the diagnosis of IUGR.

Greater flowering and response to flooding in Lythrum virgatum than L. salicaria (purple loosestrife).

Newly introduced trait diversity can spur rapid evolution and facilitate local adaptation in the introduced plant Lythrum salicaria. The horticultural plant L. virgatum might further introduce meaningful trait variation by escaping into established L. salicaria populations or by hybridizing with L. salicaria. Although many experiments have focused on L. salicaria genotypes, relatively little is known about L. virgatum ecology. We used a greenhouse common garden to compare traits and flood response of L. salicaria and L. virgatum collected from two sources each in their native range. We tested the hypotheses that these two wetland taxa have comparable responses to flooding (inundation), and that flood tolerance correlated to higher fitness. Flooding produced stronger stress responses in L. virgatum. Compared to L. salicaria, L. virgatum shifted more aboveground allocation away from reproduction, decreased inflorescence biomass by 40% more, and produced 7% more stem aerenchymatous phellum, a specialized tissue that maintains aeration. Despite these more pronounced responses to flooding stress, L. virgatum had higher fitness (inflorescence biomass and reproductive allocation) than L. salicaria. Overall, L. virgatum differed from L. salicaria in functionally important ways. Lythrum virgatum persisted under flooding and produced more reproductive biomass than L. salicaria under both flooded and non-flooded conditions. However, inundation stressed L. virgatum more than L. salicaria. Lythrum virgatum is likely able to establish into the wetland habitats in which L. salicaria prevails but may possess broader habitat tolerances.

Role of vitamin D on gestational hypertension, diabetes mellitus, timing and mode of delivery.

OBJECTIVE: To determine the efficacy of VD in preventing the development of gestational diabetes mellitus (GDM) and pregnancy-induced hypertension (PIH). The secondary purpose is to investigate the effect of VD on the mode and time of delivery. PATIENTS AND METHODS: A vitamin D value of <20 ng/mL during pregnancy is considered a deficiency according to the Endocrine Society, and 400-600 IU/day VD replacement is recommended. Forty patients whose serum VD levels were below 20 ng/mL during routine pregnancy follow-up and who were planned for VD replacement therapy were included in the study. They were divided into two equal groups with 20 patients in each group. Twenty pregnant women with serum VD levels greater than 20 ng/mL were considered as the control group. While 400 IU/day VD replacement was applied to the patients in Group 1, 600 IU/day VD was given to Group 2. Group 3 consisted of control patients who did not undergo VD replacement. VD replacement was continued from the 14th week of pregnancy until delivery. Each group of participants was screened with a 50-g GCT at 24-28 weeks of gestation. Following 50-g GCT if serum glucose level was found >140 mg/dL, patients underwent 100-g OGTT. GDM was diagnosed in the presence of at least two of the following results: fasting serum glucose ≥92 mg/dL and/or 1-hour glycemia ≥180 mg/dL, and/or 2-hour glycemia ≥153 mg/dL. PIH was defined as systolic blood pressure >140 mmHg and diastolic blood pressure >90 mmHg. Patients in each group delivered by cesarean section or normal vaginal route. In addition to the incidence of PIH and GDM, the time and mode of delivery were recorded. RESULTS: PIH was detected in two patients in each of the 400 IU/day and 600 IU/day vitamin D replacement groups (10%). In the control group, PIH developed in 3 patients (15%). Although PIH was detected in an extra case in the control group, no significant difference was found between the replacement group and the control group in terms of PIH (p<0.44). While GDM was not detected in the 400 IU/day vitamin D group, GDM was detected in one patient (5%) in the 600 IU/day vitamin D group. No case of GDM was found in the control group either. There was no significant difference between the VD replacement and the control groups in terms of GDM rates. No significant difference was found between the VD replacement and the control groups in terms of mode of delivery. While the C/S ratio was 65% in the 400 IU/day vitamin D group, this ratio was 75% in the 600 IU/day vitamin D group. There was an insignificant trend of increase in C/S ratios in the group given 600 IU/day of vitamin D. The C/S ratio of the control group, which could not be given VD replacement, was found to be 70%. CONCLUSIONS: VD replacement therapy during pregnancy does not prevent the development of PIH and GDM, and does not significantly contribute to the time and mode of delivery.

Combining In Vitro and In Silico New Approach Methods to Investigate Type 3 Iodothyronine Deiodinase Chemical Inhibition Across Species.

New approach methodologies (NAMs) are being developed to reduce and replace vertebrate animal testing in support of ecotoxicology and risk assessment. The US Environmental Protection Agency's Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) bioinformatic tool was used to evaluate amino acid sequence conservation of the type 3 iodothyronine deiodinase (DIO3) enzyme across species to demonstrate NAM applications for understanding effects of chemical interactions with a specific protein target. Existing literature was used to identify critical amino acids for thyroid hormone binding and interaction with a reducing cofactor. The SeqAPASS tool identifies whether known critical amino acids involved in ligand binding are exact, partial, or not matches across species compared with a template species based on molecular weight and side chain classification. This evaluation guided the design of variant proteins representing critical amino acid substitutions found in various species. Site-directed mutagenesis of the wild-type (WT) human DIO3 gene sequence was used to create six variant proteins expressed in cell culture, which were then tested in vitro for chemical inhibition. Significant differences in in vitro median inhibitory concentration results were observed among variants for potential competitive inhibitors. A molecular model representing the WT human DIO3 was constructed using Molecular Operating Environment (MOE) software and mutated in silico to create the six variants. The MOE Site Finder tool identified the proposed catalytic and cofactor sites and potential alternative binding sites. Virtual docking did not provide affinity scores with sufficient resolution to rank the potency of the chemical inhibitors. Chemical characteristics, function and location of substituted amino acids, and complexities of the protein target are important considerations in developing NAMs to evaluate chemical susceptibility across species. Environ Toxicol Chem 2023;42:1032-1048. © 2023 University of Wisconsin-Madison. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Autopolyploids of Arabidopsis thaliana are more phenotypically plastic than their diploid progenitors.

BACKGROUND AND AIMS: Polyploids are often hypothesized to have increased phenotypic plasticity compared with their diploid progenitors, but recent work suggests that the relationship between whole-genome duplication (WGD) and plasticity is not so straightforward. Impacts of WGD on plasticity are moderated by other evolutionary processes in nature, which has impeded generalizations regarding the effects of WGD alone. We assessed shifts in phenotypic plasticity and mean trait values accompanying WGD, as well as the adaptive consequences of these shifts. METHODS: To isolate WGD effects, we compared two diploid lineages of Arabidopsis thaliana wiht corresponding autotetraploids grown across different salt and nutrient conditions in a growth chamber. KEY RESULTS: For the few cases in which diploids and polyploids differed in plasticity, polyploids were more plastic, consistent with hypotheses that WGD increases plasticity. Under stress, increased plasticity was often adaptive (associated with higher total seed mass), but in other cases plasticity was unrelated to fitness. Mean trait values and plasticity were equally likely to be affected by WGD, but the adaptive consequences of these shifts were often context dependent or lineage specific. For example, polyploids had extended life spans, a shift that was adaptive in one polyploid lineage under amenable conditions but was maladaptive in the other lineage under stress. CONCLUSIONS: Our work shows that increased phenotypic plasticity can result from WGD alone, independent of other evolutionary processes. We find that the effects of WGD can differ depending on the genotype of the progenitor and the environmental context. Though our experiment was limited to two genotypes of a single species, these findings support the idea that WGD can indeed increase plasticity.

From Protein Sequence to Structure: The Next Frontier in Cross-Species Extrapolation for Chemical Safety Evaluations.

Computational screening for potentially bioactive molecules using advanced molecular modeling approaches including molecular docking and molecular dynamic simulation is mainstream in certain fields like drug discovery. Significant advances in computationally predicting protein structures from sequence information have also expanded the availability of structures for nonmodel species. Therefore, the objective of the present study was to develop an analysis pipeline to harness the power of these bioinformatics approaches for cross-species extrapolation for evaluating chemical safety. The Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool compares protein-sequence similarity across species for conservation of known chemical targets, providing an initial line of evidence for extrapolation of toxicity knowledge. However, with the development of structural models from tools like the Iterative Threading ASSEmbly Refinement (ITASSER), analyses of protein structural conservation can be included to add further lines of evidence and generate protein models across species. Models generated through such a pipeline could then be used for advanced molecular modeling approaches in the context of species extrapolation. Two case examples illustrating this pipeline from SeqAPASS sequences to I-TASSER-generated protein structures were created for human liver fatty acid-binding protein (LFABP) and androgen receptor (AR). Ninety-nine LFABP and 268 AR protein models representing diverse species were generated and analyzed for conservation using template modeling (TM)-align. The results from the structural comparisons were in line with the sequence-based SeqAPASS workflow, adding further evidence of LFABL and AR conservation across vertebrate species. The present study lays the foundation for expanding the capabilities of the web-based SeqAPASS tool to include structural comparisons for species extrapolation, facilitating more rapid and efficient toxicological assessments among species with limited or no existing toxicity data. Environ Toxicol Chem 2023;42:463-474. © 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Impact of spontaneous preterm birth on amniotic fluid NF-κB, IL-6, TNF-α and IL-1ß levels in singleton pregnancies conceived after IVF/ICSI treatment or natural conception.

OBJECTIVE: The aim of our study was to compare the amniotic fluid NF-κB, TNF-α, IL-1ß and IL-6 levels of patients who developed spontaneous preterm birth (sPTB) after IVF/ICSI or natural pregnancy, among themselves and with the pregnant women who gave term birth. PATIENTS AND METHODS: A total of 43 patients who had spontaneous preterm birth before 37 weeks were included in the study. While 23 out of 43 patients conceived after IVF/ICSI, the remaining 20 patients conceived spontaneously. Women in both participant groups delivered by cesarean section or vaginally. Ten patients who did not have a history of preterm labor were accepted as the control group. Amniotic fluid was taken with the aid of a 10 cc injector following spontaneous or artificial rupture of membranes from patients who presented with spontaneous preterm labor with intact membranes and started normal labor. Samples of amniotic fluid accumulated in the speculum were collected from patients with ruptured membranes at the first admission. Amniotic fluid was collected with the help of an injector just before the amniotic membrane was cut in patients who decided to have a cesarean section. NF-κB, IL-6, TNF-α and IL-1ß concentrations in amniotic fluid samples were measured quantitatively by enzyme-linked immunosorbent assay (ELISA) using human NF-κB, IL-6, TNF-α, and IL-1ß ELISA kits. RESULTS: The maternal age, parity and gestational age at the time of delivery, fetal birth weight were similar in the IVF/ICSI and natural conception groups. The amniotic fluid NF-κB, TNF-α, IL-1ß and IL-6 levels of sPTB patients in the IVF/ICSI group and those in the natural conception group were found to be similar. The tendency to increase in cytokine levels in term pregnant women compared to sPTB groups did not reach significance. Amniotic fluid proinflammatory cytokine levels of sPTB patients in both natural conception and IVF/ICSI groups were found to be similar to healthy controls with term delivery. Amniotic fluid proinflammatory cytokine levels of sPTB patients in both natural conception and IVF/ICSI groups were found to be similar to healthy controls with term delivery. There was no difference between the amniotic fluid proinflammatory cytokine levels of the patients who delivered vaginally or by cesarean section. CONCLUSIONS: Whether sPTB develops after ICSI or after natural conception, the mechanism is the same and largely overlaps with the term birth mechanism.

The effect of Nateglinide and Octreotide on follicular morphology and free radical scavenging system in letrazole-induced rat model of PCOS.

OBJECTIVE: To investigate the effects of octreotide and nateglinide on ovarian follicle count, ovarian tissue damage, biochemical parameters and free radical scavenging system in letrazole-induced rat model of PCOS. MATERIALS AND METHODS: Forty-two female Sprague-Dawley rats were divided into six groups. Group 1 (Control Group): after localizing the ovaries and the uterine horns, the abdominal wall was closed without any surgical procedure. Group 2 (PCOS Group): PCOS was induced by administrating Letrozole orally for 21 successive days. At the end of 21 days, rats underwent ovarian biopsies. The experimental PCOS model was considered successful in the presence of atretic follicles without granulosa cell stratification. Group 3 (PCOS + Nateglinide Group): Nateglinide was administered by oral dropper for 30 days to the rats in which PCOS model was created. Group 4 (Nateglinid only Group): 30 days of NG was applied to the rats without PCOS. Group 5 (PCOS+Octreotide Group): 0.1 mg/kg/day Octreotide was given intraperitoneally for 4 weeks to the rats in which PCOS model was created. Group 6 (Octreotide only Group): animals without PCOS given 0.1 mg/kg/day Octreotide at the end of the treatment, bilateral oophorectomy was performed and blood samples were collected from all groups. Ovarian tissue was stained immunohistochemically with TLR-4 in addition to conventional staining. In addition to follicle classification, ovarian damage was graded. Serum insulin, FSH and LH, TNF-α, IL-6, SHBG, SOD, IGF-1, MDA and GSH levels were also measured. RESULTS: The cystic and degenerated follicle density of PCOS group was high compared with the other groups. Both cystic and degenerated follicles were significantly reduced in PCOS+NG and PCOS+OC groups compared to PCOS group. There was no difference between the groups in terms of serum LH, FSH and insulin levels (p>0.05). Serum testosterone level was significantly higher in the PCOS group compared to the other groups (p<0.01). Adding OC or NG to PCOS groups did not cause significant changes in testosterone levels. TNF-α and IL-6 levels were high in PCOS group (p<0.03). IGF-1 and MDA levels were higher in PCOS than in other groups (p<0.03, p<0.01 respectively). Adding OC or NG to the treatment normalized IGF-1 and MDA levels. Serum GSH levels were significantly lower in the PCOS group (p<0.05). Adding NG to the treatment increased GSH levels. CONCLUSIONS: Both NG and OCT reverses atretic and degenerate follicle damage due to PCOS through TLR-4, antioxidant and anti-inflammatory pathways.

Fertility protective effect of taxifolin in cisplatin-induced ovarian damage.

OBJECTIVE: The aim of our study was to investigate the protective effect of taxifolin on ovarian damage and reproductive dysfunction created by cisplatin administration. MATERIALS AND METHODS: A total of 36 albino Wistar female adult rats were equally divided into 3 groups as cisplatin administered only (CIS), taxifolin+cisplatin (T+C) and healthy control group (HG). Taxifolin 50 mg/kg was administered orally by gavage in the T+C (n=12) group. In the HG (n=12) and CIS (n=12) groups, the same volume of distilled water as a solvent was orally administered. One hour after administration of taxifolin or distilled water, animals in the T+C and CIS groups were injected with cisplatin at a dose of 2.5 mg/kg intraperitoneally. This procedure was repeated once a day for 14 days. Six animals from each group were sacrificed on day 15, and their ovaries were removed for histopathological and biochemical analysis. Ovarian tissue malondialdehyde (MDA), total Glutathione (tGSH), Nuclear Factor-Kappa B (NF-kB), Tumor Necrosis Factor-α (TNF-α), Interleukin 1 beta (IL-1ß), and Interleukin-6 (IL-6) levels were measured. The remaining animals (n=6 in each group) were kept in the laboratory with mature male rats for two months to breed. RESULTS: CIS administration led to an increase in inflammatory molecules and membrane lipid peroxidation products, and decreased the synthesis of antioxidant molecules. Compared to the CIS group, the ovarian tissue MDA, NF-kB, TNF-α, IL-1ß and IL-6 levels were found to be significantly decreased in the T+C group (p<0.001 for all comparisons). On the other hand, the tGSH levels of the T+C group were significantly higher than the CIS group (p<0.001). Milder ovarian necrosis, fibrosis and follicle damage were detected in animals which were given taxifolin. Four out of the six rats (67%) treated with taxifolin gave birth within 27 days. CONCLUSIONS: We demonstrated, for the first time, that taxifolin ameliorates cisplatin-induced ovarian injury by decreasing MDA and proinflammatory cytokines and increasing the antioxidant enzyme. The fact that more than half of the animals receiving taxifolin became pregnant suggests that the cytoprotective effect of taxifolin is strong enough to preserve fertility.

DNA repair gene (XRCC1 and XPD) polymorphism and risk of primary ovarian failure.

OBJECTIVE: DNA repair genes may be related with the onset of primary ovarian failure (POF). The study was planned to investigate whether the polymorphisms in the DNA repair genes modulate the risk of POF. PATIENTS AND METHODS: This prospective study included 25 women diagnosed with POF and 25 healthy controls. The genotyping and allele of XRCC1 and XPD genes were determined by using Polymerase Chain Reaction and fluorescence melting curve analysis. RESULTS: The genotype and allele distribution of the Arg194Trp and Arg399Lys polymorphism of the XRCC1 gene did not differ statistically between those of the POF and control groups. The frequency of the C (Gln) allele was significantly lower in patients diagnosed with POF when compared to that in healthy controls [48% vs. 76%, p=0.040, OR: 3.43 (CI: 1.03-11.48)]. The Lys/Lys genotype for XPD-Lys751Gln polymorphism in patients diagnosed with POF was more common than in healthy controls (p=0.028, 52% vs. 24%). CONCLUSIONS: The genotype distribution and allele frequency of XPD-Lys751Gln, XRCC1-Arg194Trp and XRCC1-Arg399 Gln did not regulate the risk of developing POF. Gln/Gln+Lys/Gln and XPD-Lys751Gln polymorphism may have a possible protective role against the development of POF.

Comparison of immunohistochemical characteristics of endometriomas with non-endometriotic benign ovarian cysts.

OBJECTIVE: The aim of the study was to investigate the existence of neuroendocrine cells and to compare the density of those in normal ovarian tissue, endometriotic and non-endometriotic benign ovarian cysts. PATIENTS AND METHODS: Twenty patients with the diagnosis of endometrioma and 30 control subjects consisting of ovarian serous cystadenoma (n=10), ovarian mucinous cystadenoma (n=10) and normal ovarian tissue (n=10) were included. The tissues were prepared and assessed according to staining density by using the H-score method. RESULTS: Tissues with mucinous cystadenoma were significantly more stained with PAS and VanGieson, when compared to women with endometrioma. Macrophage deposition was higher in cyst samples with endometrioma and in normal ovarian tissue when compared to serous cystadenoma and mucinous cystadenoma. Normal ovarian tissue was significantly more stained with PGP9.5, NSE and SYN when compared to endometrioma and non-endometriotic benign ovarian cyst. PGP9.5 staining was higher in normal ovarian tissue when compared with endometriotic lesions (p<.001). Endometrioma samples were significantly more stained with p53 when compared to non-endometriotic cysts and normal ovarian tissue. c-Kit staining was mild and not statistically significant among all groups. CONCLUSIONS: During endometrioma transformation, expression intensity of neuroendocrine markers decreases compared to normal ovarian tissue and other benign ovarian cysts.

Surgical removal of intramural fibroids improves the TNF-α induced inflammatory events in endometrium.

OBJECTIVE: To investigate whether surgical removal of intramural fibroids makes any changes in the expression of endometrial tumor necrosis factor alpha (TNF-α) and nuclear factor kappa B (NF-κB). PATIENTS AND METHODS: 20 patients who had no pathology other than intramural fibroid as a cause of subfertility were included in the study. 6 patients who planned tubal ligation were considered as the control group. Type 3 or 4 fibroid diagnosis was made according to the revised International Federation of Gynecology and Obstetrics (FIGO). At the end of the diagnostic and confirmatory tests, 8 patients were diagnosed with type 3 and 12 patients with type 4 fibroids. While the patients in the fibroid group went to myomectomy, the patients in the control group went to tubal ligation. Before the myomectomy, endometrial sampling was performed with a pipelle cannula under anesthesia. The same procedure was repeated three months after the treatment. The same endometrial sampling procedure was also performed on the patients in the control group. TNF-alpha and NF-κB levels were measured with enzyme-linked immunosorbent assay (ELISA) in endometrial samples taken before and three months after myomectomy. RESULTS: Pre-myomectomy TNF-α levels of the patients in the type 3 fibroid group were significantly higher than the control group (5.10±1.30 vs. 2.50±0.11, p<0.01). Similarly, the pre-myomectomy TNF-α levels of the patients in the type 4 fibroid group were significantly higher than the control group (4.73±1.76 vs. 2.50±0.11, p<0.01). There was no significant difference between pre-myomectomy endometrial TNF-α levels of patients in type 3 and 4 fibroid groups (5.10±1.30 vs. 4.73±1.76, p>0.05). Removal of type 3 fibroids by myomectomy significantly decreased TNF-α levels (5.10±1.30 vs. 2.20±0.44, p<0.03). Similarly, removal of type 4 fibroids by myomectomy significantly decreased TNF-α levels (4.73±1.76 vs. 2.60±0.30, p<0.04). Pre-myomectomy NF-κB levels of the patients in the type 3 fibroid group were significantly higher than the control group (2.42±0.39 vs. 1.09±0.60, p<0.02). Similarly, the pre-myomectomy NF-κB levels of the patients in the type 4 fibroid group were significantly higher than the control group (2.04±0.50 vs. 1.09±0.60, p<0.01). There was no significant difference between the pre-myomectomy endometrial NF-κB levels of the patients in the type 3 and 4 fibroid groups (2.42±0.39 vs. 2.04±0.50 p>0.05). Removal of type 3 fibroids by myomectomy significantly decreased NF-κB levels (2.42±0.39 vs. 1.02±0.33, p<0.01). Similarly, removal of type 4 fibroids by myomectomy significantly decreased NF-κB levels (2.04±0.50 vs. 0.97±0.02, p<0.02). CONCLUSIONS: Surgical removal of type 3 or type 4 fibroids contributes positively to receptivity by down-regulating endometrial TNF-α and NF-ƙB.

Potential local adaptation in populations of invasive reed canary grass (Phalaris arundinacea) across an urbanization gradient.

Urban stressors represent strong selective gradients that can elicit evolutionary change, especially in non-native species that may harbor substantial within-population variability. To test whether urban stressors drive phenotypic differentiation and influence local adaptation, we compared stress responses of populations of a ubiquitous invader, reed canary grass (Phalaris arundinacea). Specifically, we quantified responses to salt, copper, and zinc additions by reed canary grass collected from four populations spanning an urbanization gradient (natural, rural, moderate urban, and intense urban). We measured ten phenotypic traits and trait plasticities, because reed canary grass is known to be highly plastic and because plasticity may enhance invasion success. We tested the following hypotheses: (a) Source populations vary systematically in their stress response, with the intense urban population least sensitive and the natural population most sensitive, and (b) plastic responses are adaptive under stressful conditions. We found clear trait variation among populations, with the greatest divergence in traits and trait plasticities between the natural and intense urban populations. The intense urban population showed stress tolerator characteristics for resource acquisition traits including leaf dry matter content and specific root length. Trait plasticity varied among populations for over half the traits measured, highlighting that plasticity differences were as common as trait differences. Plasticity in root mass ratio and specific root length were adaptive in some contexts, suggesting that natural selection by anthropogenic stressors may have contributed to root trait differences. Reed canary grass populations in highly urbanized wetlands may therefore be evolving enhanced tolerance to urban stressors, suggesting a mechanism by which invasive species may proliferate across urban wetland systems generally.