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Objective: To study the effects of n-6 and n-3 polyunsaturated acid-rich soybean phosphatidylcholine (soy-PC) on sperm cryotolerance with regard to sperm membrane lipid profile, membrane surface integrity, and routine semen parameters., Design: Experimental study., Setting: University-affiliated tertiary hospital., Patient(s): A total of 20 normospermic fertile men., Intervention(s): Semen samples examined for differences in semen parameters, sperm membrane lipid profile, and plasma membrane surface both before and after cryopreservation using basic freezing medium with N-tris(hydroxymethyl)-methyl-2-aminoethane sulfonic acid (TES) and tris-(hydroxymethyl)-aminomethane (TRIS) supplemented with purified soy-PC (TEST-PC) or egg yolk (TEST-Y), both alone or in association (TEST-Y-PC)., Main Outcome Measure(s): Conventional semen parameters and membrane lipid profile by matrix-assisted laser/desorption ionization mass spectrometry (MALDI-MS)., Result(s): Postthaw sperm cell motility, vitality, and morphology parameters were similar for soy-PC (TEST-PC) and egg yolk (TEST-Y) cryoprotectants. However, sperm exposed to TEST-Y-PC presented better kinetic parameters, which were similar to the original quality of the fresh semen. Human sperm MALDI-MS lipid profiles revealed that the relative abundance of glycerophospholipids of m/z 760.44 [PC (34:1)+H]+, 781.55 [SM (20:0) +Na]+, 784.55 [PC (36:3) +H]+, 806.64 [PC (38:6) +H]+, 807.64 [SM (22:1) +Na]+, and 809.64 [SM (22:0) +Na]+ increased in soy-PC samples (TEST-PC). Nonetheless, only one lipid (m/z 781.55, [SM (20:0) +Na]+) statistically significantly changed when sperm was cryopreserved in TEST-Y-PC., Conclusion(s): Sphingomyelin was defined as a prospective biomarker of soy-PC treatment, and it could be related to the positive cryoprotective effects of soy-PC in human sperm, opening new perspectives to design of a more efficient synthetic cryoprotectant medium containing purified egg yolk biomolecules combined with soy-PC., (Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Mammalian preimplantation embryonic development is a complex, conserved, and well-orchestrated process involving dynamic molecular and structural changes. Understanding membrane lipid profile fluctuation during this crucial period is fundamental to address mechanisms governing embryogenesis. Therefore, the aim of the present work was to perform a comprehensive assessment of stage-specific lipid profiles during early bovine embryonic development and associate with the mRNA abundance of lipid metabolism-related genes (ACSL3, ELOVL5, and ELOVL6) and with the amount of cytoplasmic lipid droplets. Immature oocytes were recovered from slaughterhouse-derived ovaries, two-cell embryos, and eight- to 16-cell embryos, morula, and blastocysts that were in vitro produced under different environmental conditions. Lipid droplets content and mRNA transcript levels for ACSL3, ELOVL5, and ELOVL6, monitored by lipid staining and quantitative polymerase chain reaction, respectively, increased at morula followed by a decrease at blastocyst stage. Relative mRNA abundance changes of ACSL3 were closely related to cytoplasmic lipid droplet accumulation. Characteristic dynamic changes of phospholipid profiles were observed during early embryo development and related to unsaturation level, acyl chain length, and class composition. ELOVL5 and ELOVL6 mRNA levels were suggestive of overexpression of membrane phospholipids containing elongated fatty acids with 16, 18, and 20 carbons. In addition, putative biomarkers of key events of embryogenesis, embryo lipid accumulation, and elongation were identified. This study provides a comprehensive description of stage-specific lipidome signatures and proposes a mechanism to explain its potential relationship with the fluctuation of both cytoplasmic lipid droplets content and mRNA levels of lipid metabolism-related genes during early bovine embryo development., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The zebrafish Danio rerio is a model vertebrate organism for understanding biological mechanisms. Recent studies have explored using zebrafish as a model for lipid-related diseases, for in vivo fish bioassays, and for embryonic toxicity experiments. Mass spectrometry (MS) and MS imaging are established tools for lipid profiling and spatial mapping of biomolecules and offer rapid, sensitive, and simple analytical protocols for zebrafish analysis. When ambient ionization techniques are used, ions are generated in native environmental conditions, requiring neither sample preparation nor separation of molecules prior to MS. We used two direct MS techniques to describe the dynamics of the lipid profile during zebrafish embryonic development from 0 to 96 hours post-fertilization and to explore these analytical approaches as molecular diagnostic assays. Desorption electrospray ionization (DESI) MS imaging followed by nanoelectrospray (nESI) MS and tandem MS (MS/MS) were used in positive and negative ion modes, allowing the detection of a large variety of phosphatidylglycerols, phosphatidylcholines, phosphatidylinositols, free fatty acids, triacylglycerols, ubiquinone, squalene, and other lipids, and revealed information on the spatial distributions of lipids within the embryo and on lipid molecular structure. Differences were observed in the relative ion abundances of free fatty acids, triacylglycerols, and ubiquinone - essentially localized to the yolk - across developmental stages, whereas no relevant differences were found in the distribution of complex membrane glycerophospholipids, indicating conserved lipid constitution. Embryos exposed to trichloroethylene for 72 hours exhibited an altered lipid profile, indicating the potential utility of this technique for testing the effects of environmental contaminants.

Mass spectrometry imaging is a powerful tool for investigating the spatial distribution of chemical compounds in a biological sample such as tissue. Two common goals of these experiments are unsupervised segmentation of images into newly discovered homogeneous segments and supervised classification of images into predefined classes. In both cases, the important secondary goals are to characterize the uncertainty associated with the segmentation and with the classification and to characterize the spectral features that define each segment or class. Recent analysis methods have focused on the spatial structure of the data to improve results. However, they either do not address these secondary goals or do this with separate post hoc procedures.We introduce spatial shrunken centroids, a statistical model-based framework for both supervised classification and unsupervised segmentation. It takes as input sets of previously detected, aligned, quantified, and normalized spectral features and expresses both spatial and multivariate nature of the data using probabilistic modeling. It selects informative subsets of spectral features that define each unsupervised segment or supervised class and quantifies and visualizes the uncertainty in spatial segmentations and in tissue classification. In the unsupervised setting, it also guides the choice of an appropriate number of segments. We demonstrate the usefulness of this framework in a supervised human renal cell carcinoma experimental dataset and several unsupervised experimental datasets, including a pig fetus cross-section, three rodent brains, and a controlled image with known ground truth. This framework is available for use within the open-source R package Cardinal as part of a full pipeline for the processing, visualization, and statistical analysis of mass spectrometry imaging experiments., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Objectives: The aim of this study was to evaluate the impact of a bundle called FAST HUG in ventilator-associated pneumonia, weigh the healthcare costs of ventilator-associated pneumonia patients in the intensive care unit, and hospital mortality due to ventilator-associated pneumonia., Material and Methods: The study was performed in a private hospital that has an 8-bed intensive care unit. It was divided into two phases: before implementing FAST HUG, from August 2011 to August 2012 and after the implementation of FAST HUG, from September 2012 to December 2013. An individual form for each patient in the study was filled out by using information taken electronically from the hospital medical records. The following data was obtained from each patient: age, gender, reason for hospitalization, use of three or more antibiotics, length of stay, intubation time, and outcome., Results: After the implementation of FAST HUG, there was an observable decrease in the occurrence of ventilator-associated pneumonia (p<0.01), as well as a reduction in mortality rates (p<0.01). In addition, the intervention resulted in a significant reduction in intensive care unit hospital costs (p<0.05)., Conclusion: The implementation of FAST HUG reduced the number of ventilator-associated pneumonia cases. Thus, decreasing costs, reducing mortality rates and length of stay, which therefore resulted in an improvement to the overall quality of care., (Copyright © 2016 Elsevier Editora Ltda. All rights reserved.)

Patients with generalized arterial calcification of infancy (GACI) develop vascular calcifications early in life. About half of them die within the first 6 months despite optimal medical care. A subset of those who survive eventually develop hypophosphatemic rickets. Since hypophosphatemia and hyperphosphaturia have been previously associated with increased survival in GACI patients, physicians often avoid phosphate repletion as treatment for rickets. As a consequence, GACI patients develop severe rachitic complications such as short stature and skeletal deformities. It appears that the recognition of hypophosphatemia later in life in some GACI patients is a consequence of having survived the first few months of life, and not the cause of their survival per se. Here, we report the long-term follow-up of a GACI patient who was phosphate-repleted for his rickets for more than 7 years without worsening of vascular calcification., (© 2016 Wiley Periodicals, Inc.)

Polybromo-1 (PBRM1) is a component of the PBAF (Polybromo-associated-BRG1- or BRM-associated factors) chromatin remodeling complex and is the second most frequently mutated gene in clear-cell renal cell Carcinoma (ccRCC). Mutation of PBRM1 is believed to be an early event in carcinogenesis, however its function as a tumor suppressor is not understood. In this study, we have employed Next Generation Sequencing to profile the differentially expressed genes upon PBRM1 re-expression in a cellular model of ccRCC. PBRM1 re-expression led to upregulation of genes involved in cellular adhesion, carbohydrate metabolism, apoptotic process and response to hypoxia, and a downregulation of genes involved in different stages of cell division. The decrease in cellular proliferation upon PBRM1 re-expression was confirmed, validating the functional role of PBRM1 as a tumor suppressor in a cell-based model. In addition, we identified a role for PBRM1 in regulating metabolic pathways known to be important for driving ccRCC, including the regulation of hypoxia response genes, PI3K signaling, glucose uptake, and cholesterol homeostasis. Of particular novelty is the identification of cell adhesion as a major downstream process uniquely regulated by PBRM1 expression. Cytoskeletal reorganization was induced upon PBRM1 reexpression as evidenced from the increase in the number of cells displaying cortical actin, a hallmark of epithelial cells. Genes involved in cell adhesion featured prominently in our transcriptional dataset and overlapped with genes uniquely regulated by PBRM1 in clinical specimens of ccRCC. Genes involved in cell adhesion serve as tumor suppressor and maybe involved in inhibiting cell migration. Here we report for the first time genes linked to cell adhesion serve as downstream targets of PBRM1, and hope to lay the foundation of future studies focusing on the role of chromatin remodelers in bringing about these alterations during malignancies.

Purpose: The purpose of this study is to verify the use of medicinal plants by pregnant women treated at four Basic Health Units and at a public maternity facility in Brazil's northeast., Methods: This is a cross-sectional, quantitative study, performed between February and April 2014. The subjects were 178 pregnant women, aged 18 to 42 years. To collect data, a structured questionnaire with dichotomous and multiple choice questions was used. To verify the correlation between the variables, Pearson's chi-square test was used., Results: The study showed that 30.9% of the pregnant women used medicinal plants, and boldo was the most cited (35.4%). All the plants utilized, except lemongrass, have toxic effects in pregnancy, according to Resolution SES/RJ N° 1757. There was no statistically significant correlation between social class and use of medicinal plants., Conclusion: The health of the study participants and their unborn children is at risk due to the inappropriate use of medicinal plants., (Thieme Publicações Ltda Rio de Janeiro, Brazil.)

Background: One driving motivation in the development of point-of-care (POC) diagnostics is to conveniently and immediately provide information upon which healthcare decisions can be based, while the patient is on site. Ambient ionization mass spectrometry (MS) allows direct chemical analysis of unmodified and complex biological samples. This suite of ionization techniques was introduced a decade ago and now includes a number of techniques, all seeking to minimize or eliminate sample preparation. Such approaches provide new opportunities for POC diagnostics and rapid measurements of exogenous and endogenous molecules (e.g., drugs, proteins, hormones) in small volumes of biological samples, especially when coupled with miniature mass spectrometers., Content: Ambient MS-based techniques are applied in diverse fields such as forensics, pharmaceutical development, reaction monitoring, and food analysis. Clinical applications of ambient MS are at an early stage but show promise for POC diagnostics. This review provides a brief overview of various ambient ionization techniques providing background, examples of applications, and the current state of translation to clinical practice. The primary focus is on paper spray (PS) ionization, which allows quantification of analytes in complex biofluids. Current developments in the miniaturization of mass spectrometers are discussed., Summary: Ambient ionization MS is an emerging technology in analytical and clinical chemistry. With appropriate MS instrumentation and user-friendly interfaces for automated analysis, ambient ionization techniques can provide quantitative POC measurements. Most significantly, the implementation of PS could improve the quality and lower the cost of POC testing in a variety of clinical settings., (© 2015 American Association for Clinical Chemistry.)

The majority of lipids in the brain are located in the bilayer membranes. These lipids are not visible by magnetic resonance spectroscopy since they have restricted mobility. Only mobile lipids, such as cholesterol esters or triglycerides in neutral lipid droplets, have enough rotational freedom to generate a signal on spectroscopy. These signals are detected as peaks at 1.3 ppm, originating from the methylene groups in the fatty acid chain, and 0.9 ppm, originating from the distal methyl group. We review the literature on the different genetic conditions that have been found to show lipid peaks on brain spectroscopy and report the first patient with carnitine palmitoyltransferase 2 deficiency shown to have such lipid peaks, thus indicating brain fat accumulation.

Streptococcus agalactiae is a well-known pathogen during pregnancy and in neonates. Among non-pregnant adults, invasive infection, although rare, is showing increasing frequency, especially in chronically ill, immunosuppressed, or older patients. Although rare, the clinical features of meningeal infection caused by S. agalactiae are similar to other bacterial meningitis. The authors report the case of a middle-aged man previously diagnosed with hypertension, diabetes mellitus, and alcoholic liver cirrhosis, who was admitted at the emergency department with a Glasgow Coma Scale of 11/12, generalized spasticity, bilateral Babinski sign, and hypertension. The clinical outcome was bad, with refractory shock and death within 24 hours of hospitalization. The bacteriological work-up isolated S. agalactiae in the cerebral spinal fluid (CSF), blood, and urine. An autopsy revealed meningoencephalitis, acute myocardial infarction, and pyelonephritis due to septic emboli. The authors point out the atypical CSF findings, the rapid fatal outcome, and the importance of including this pathogen among the etiologic possibilities of invasive infections in this group of patients.

Identifying factors that can be related to the occurrence of gestational arterial hypertension. The sample was composed by 105 pregnant women in their third trimester of gestation, during the period between September 2013 and August 2014. General assessment questionnaires together with a questionnaire to evaluate anxiety (STAI-A-STATE) were applied; arterial blood pressure values were collected. To classify anxiety, a mean of the final result of all the questionnaires gotten was calculated. Pregnant women who showed scores higher than the mean were considered anxious. All data were analyzed by a logistic regression. The significance level adopted was 0.05. A data analysis allowed us to verify that 92.38% of the pregnant women had an anxious personality STAI-A-STATE and 12.38% of them had a momentary hypertension. The momentary hypertension showed a correlation between the hypertension and the state anxiety score (p = 0.049). The hypertension showed an association with the presence of depression (OR 8.69), obesity (OR 6.45), anxiety (OR 7.77), nausea (OR 12.79) and non-white race (OR 8.18). According to the study realized, the factors non-white race, depression, nausea, obesity and anxiety can be considered risk factors for the occurrence of gestational arterial hypertension. Based on these findings, a high quality prenatal assistance is considered of prime importance.

This study aimed to evaluate the impact of vitrification on membrane lipid profile obtained by mass spectrometry (MS) of in vitro-produced bovine embryos. Matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) has been used to obtain individual embryo membrane lipid profiles. Due to conditions of analysis, mainly membrane lipids, most favorably phosphatidylcholines (PCs) and sphingomyelins (SMs) have been detected. The following ions described by their mass-to-charge ratio (m/z) and respective attribution presented increased relative abundance (1.2-20×) in the vitrified group: 703.5 [SM (16:0) + H]+; 722.5 [PC (40:3) + Na]+; 758.5 [PC (34:2) + H]+; 762.5 [PC (34:0) + H]+; 790.5 [PC (36:0) + H]+ and 810.5 [PC (38:4) + H]+ and/or [PC (36:1) + Na]+. The ion with a m/z 744.5 [PCp (34:1) and/or PCe (34:2)] was 3.4-fold more abundant in the fresh group. Interestingly, ions with m/z 722.5 or 744.5 indicate the presence of lipid species, which are more resistant to enzymatic degradation as they contain fatty acyl residues linked through ether type bonds (alkyl ether or plasmalogens, indicated by the lowercase 'e' and 'p', respectively) to the glycerol structure. The results indicate that cryopreservation impacts the membrane lipid profile, and that these alterations can be properly monitored by MALDI-MS. Membrane lipids can therefore be evaluated by MALDI-MS to monitor the effect of cryopreservation on membrane lipids, and to investigate changes in lipid profile that may reflect the metabolic response to the cryopreservation stress or changes in the environmental conditions.

The aim of this study was to evaluate the effect of supplementing serum-containing media with a mixture of cis- and trans-9,11- and -10,12-conjugated isomers of linoleic acid (CLA) during different steps of the in vitro production (IVM, IVC, or IVM + IVC) of bovine embryos on their embryonic development, cryotolerance, and lipid profile. To evaluate the impact of the CLA on membrane lipids, such as phosphatidylcholine (PC) and sphingomyelin (SM), the embryos' lipid profiles were obtained using matrix-assisted laser desorption ionization mass spectrometry. The cleavage rates (78.6%-84.8%) and blastocyst development (44.8%-51.2%) remained unaltered. The postthawing reexpansion rates were higher (P < 0.05) when the CLA was added to the IVM medium (82.6%) or to the IVM + IVC medium (83.8%) than the control (69.3%) or IVC medium (63.0%). Changes in the blastocysts' lipid profile occurred when supplementation was restricted to the IVM or IVC medium. However, the most prominent effects of the CLA on the embryonic PC and SM profiles were observed when the supplement was added to IVM + IVC media, which was an increase in the level of highly unsaturated PCs containing 36 or 38 carbons, which are likely to contain CLA residues. These results showed that the molecular mechanism resulting in the improved cryosurvival, observed with CLA supplementation during bovine embryonic in vitro production, was related to the composition of structural lipids of cellular membranes and is dependent on the treatment length. Monitoring the lipid profile of embryonic membranes may improve the CLA supplementation strategy and facilitate the development of new IVC systems to improve the cryopreservation of bovine embryos and those of other domestic species., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Purpose: To report the experience and the outcomes of interstitial high-dose-rate (HDR) brachytherapy (BT) of eyelid skin cancer at the Department of Radiotherapy of Hospital de Santa Maria in Lisbon., Methods and Materials: Seventeen patients (pts; mean age, 73.75 years) who underwent eyelid interstitial HDR BT with an (192)Ir source between January 2011 and February 2013 were analyzed. Lesions were basal (94%) and squamous (6%) cell carcinomas, on lower (88%) or upper (6%) eyelids, and on inner canthus (6%). T-stage was Tis (6%), T1 (46%), T2 (36%), and T3a (12%). The purpose of BT was radical (12%), adjuvant to surgery (71%), or salvage after surgery (18%). The BT implant and treatment planning were based on the Stepping Source Dosimetry System. The median total dose was 42.75 Gy (range, 32-50 Gy), with a median of 10 fractions (range, 9-11 fractions), twice daily, 6 h apart. The median V100 was 2.38 cm(3) (range, 0.83-5.59 cm(3)), and the median V150 was 1.05 cm(3) (range, 0.24-3.12 cm(3))., Results: At a median followup of 40 months (range, 7-43 months), the local control was 94.1%. There was one local recurrence and one non-related death. The BT was well tolerated. Madarosis was the most common late effect (65% of pts) and was related with higher values of V100 (p = 0.027). Cosmetic outcomes were good and excellent in 70% of pts., Conclusions: Interstitial HDR BT is a feasible and safe technique for eyelid skin cancers, with good local control. Recurrent lesions and higher volumes receiving the prescribed dose were associated with worse outcomes., (Copyright © 2015 American Brachytherapy Society. Published by Elsevier Inc. All rights reserved.)

PIGT-CDG, an autosomal recessive syndromic intellectual disability disorder of glycosylphosphatidylinositol (GPI) anchors, was recently described in two independent kindreds [Multiple Congenital Anomalies-Hypotonia-Seizures Syndrome 3 (OMIM, #615398)]. PIGT encodes phosphatidylinositol-glycan biosynthesis class T, a subunit of the heteropentameric transamidase complex that facilitates the transfer of GPI to proteins. GPI facilitates attachment (anchoring) of proteins to cell membranes. We describe, at ages 7 and 6 years, two children of non-consanguineous parents; they had hypotonia, severe global developmental delay, and intractable seizures along with endocrine, ophthalmologic, skeletal, hearing, and cardiac anomalies. Exome sequencing revealed that both siblings had compound heterozygous variants in PIGT (NM&#95;015937.5), i.e., c.918dupC, a novel duplication leading to a frameshift, and c.1342C > T encoding a previously described missense variant. Flow cytometry studies showed decreased surface expression of GPI-anchored proteins on granulocytes, consistent with findings in previous cases. These siblings further delineate the clinical spectrum of PIGT-CDG, reemphasize the neuro-ophthalmologic presentation, clarify the endocrine features, and add hypermobility, low CSF albumin quotient, and hearing loss to the phenotypic spectrum. Our results emphasize that GPI anchor-related congenital disorders of glycosylation (CDGs) should be considered in subjects with early onset severe seizure disorders and dysmorphic facial features, even in the presence of a normal carbohydrate-deficient transferrin pattern and N-glycan profiling. Currently available screening for CDGs will not reliably detect this family of disorders, and our case reaffirms that the use of flow cytometry and genetic testing is essential for diagnosis in this group of disorders., (Copyright © 2015. Published by Elsevier Inc.)

Golden retriever muscular dystrophy (GRMD) provides the best animal model for characterizing the disease progress of the human disorder, Duchenne muscular dystrophy (DMD). The purpose of this study was to determine steroid hormone concentration profiles in healthy golden retriever dogs (control group - CtGR) versus GRMD-gene carrier (CaGR) and affected female dogs (AfCR). Therefore, a sensitive and specific analytical method was developed and validated to determine the estradiol, progesterone, cortisol, and testosterone levels in the canine serum by isotope dilution liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). To more accurately understand the dynamic nature of the serum steroid profile, the fluctuating levels of these four steroid hormones over the estrous cycle were compared across the three experimental groups using a multivariate statistical analysis. The concentration profiles of estradiol, cortisol, progesterone, and testosterone revealed a characteristic pattern for each studied group at each specific estrous phase. Additionally, several important changes in the serum concentrations of cortisol and estradiol in the CaGR and AfCR groups seem to be correlated with the status and progression of the muscular dystrophy. A comprehensive and quantitative monitoring of steroid profiles throughout the estrous cycle of normal and GRMD dogs were achieved. Significant differences in these profiles were observed between GRMD and healthy animals, most notably for estradiol. These findings contribute to a better understanding of both dog reproduction and the muscular dystrophy pathology. Our data open new venues for hormonal behavior studies in dystrophinopathies and that may affect the quality of life of DMD patients.

Objective: To determine the prevalence of anxiety and gestational depression and to determine the risk factors associated to its development., Method: Pregnant women during their third quarter of pregnancy were invited to participate to the study, and they signed an informed consent form. After that, they filled in a general assessment questionnaire in which socio-economical and obstetrical characteristics together with general health conditions and behaviors harmful for the pregnancy were evaluated. To determine the anxiety symptoms a STAI A-trait scale and a STAI-A-state scale were used and to measure the depression a CES-D scale was used. A data logistic regression was realized to determine significant associations. The significance level adopted was 0.05 for all statistical tests., Results: The final sample was represented by 207 pregnant women and the prevalence of depression was the highest value (73.5%), followed by state anxiety (58.5%) and finally by trait anxiety (53.2%). Only trait anxiety was explained by the variables studied and it was associated to the realization of a treatment for chronic diseases (OR = 2.93; IC = 1.02-8.41; p = 0.045) and the usage of continuous prescription drugs (OR = 2.30; IC = 1.06-4.97; p = 0.034)., Conclusions: The prevalence of anxiety and depression among pregnant women were both high and only trait anxiety was explained by treatment for chronic diseases and the usage of continuous prescription drugs.

Lipids play fundamental roles in mammalian embryo preimplantation development and cell fate. Triacylglycerol accumulates in oocytes and blastomeres as lipid droplets, phospholipids influence membrane functional properties, and essential fatty acid metabolism is important for maintaining the stemness of cells cultured in vitro. The growing impact that lipids have in the field of developmental biology makes analytical approaches to analyse structural information of great interest. This paper describes the concept and presents the results of lipid profiling by mass spectrometry (MS) of oocytes and preimplantation embryos, with special focus on ambient ionisation. Based on our previous experience with oocytes and embryos, we aim to convey that ambient MS is also valuable for stem cell differentiation analysis. Ambient ionisation MS allows the detection of a wide range of lipid classes (e.g. free fatty acids, cholesterol esters, phospholipids) in single oocytes, embryos and cell pellets, which are informative of in vitro culture impact, developmental and differentiation stages. Background on MS principles, the importance of underused MS scan modes for structural analysis of lipids, and statistical approaches used for data analysis are covered. We envisage that MS alone or in combination with other techniques will have a profound impact on the understanding of lipid metabolism, particularly in early embryo development and cell differentiation research.

Nutritional and environmental conditions around conception and during early embryonic development may have significant effects on health and well-being in adult life. Here, a bovine heifer model was used to investigate the effects of rumen-protected fat supplementation on oocyte quality and embryo development. Holstein-Friesian heifers (n=84) received a dietary supplement consisting of rumen-protected conjugated linoleic acid (CLA) or stearic acid (SA), each on top of an isocaloric basic diet. Oocytes were collected via ultrasound-guided follicular aspiration and subjected to in vitro maturation followed by either desorption electrospray ionisation mass spectrometry (DESI-MS) for lipid profiling of individual oocytes or in vitro fertilisation and embryo culture. The type of supplement significantly affected lipid profiles of in vitro-matured oocytes. Palmitic acid and plasmalogen species were more abundant in the mass spectra of in vitro-matured oocytes after rumen-protected SA supplementation when compared with those collected from animals supplemented with CLA. Lipid concentrations in blood and follicular fluid were significantly affected by both supplements. Results show that rumen-protected fatty-acid supplementation affects oocyte lipid content and may pave the way for the establishment of a large-animal model for studies towards a better understanding of reproductive disorders associated with nutritional impairments.

Some embryos exhibit better survival potential to cryopreservation than others. The cause of such a phenotype is still unclear and may be due to cell damage during cryopreservation, resulting from overaccumulation and composition of lipids. In cattle embryos, in vitro culture conditions have been shown to impact the number of lipid droplets within blastomeres. Thus far, the impact of breed on embryonic lipid content has not been studied. In the present study were compared the colour, lipid droplet abundance, lipid composition, mitochondrial activity and gene expression of in vivo-collected Jersey breed embryos, which are known to display poor performance post-freezing, with those of in vivo Holstein embryos, which have good cryotolerance. Even when housed and fed under the same conditions, Jersey embryos were found to be darker and contain more lipid droplets than Holstein embryos, and this was correlated with lower mitochondrial activity. Differential expression of genes associated with lipid metabolism and differences in lipid composition were found. These results show genetic background can impact embryonic lipid metabolism and storage.

The decreased rate of pregnancy obtained in cattle using frozen in vitro embryos compared with in vivo embryos has been associated with over-accumulation of intracellular lipid, which causes cell damage during cryopreservation. It is believed that the higher lipid content of blastomeres of bovine embryos produced in vitro results in darker-coloured cytoplasm, which could be a consequence of impaired mitochondrial function. In this study, l-carnitine was used as a treatment to reduce embryonic lipid content by increasing metabolism in cultured bovine embryos. We have observed previously that in vivo embryos of different dairy breeds collected from cows housed and fed under the same conditions differed in lipid content and metabolism. As such, breed effects between Holstein and Jersey were also examined in terms of general appearance, lipid composition, mitochondrial activity and gene expression. Adding l-carnitine to the embryo culture medium reduced the lipid content in both breeds due to increased mitochondrial activity. The response to l-carnitine was weaker in Jersey than in Holstein embryos. Our results thus show that genetics influence the response of bovine embryos to stimulation of mitochondrial metabolism.

Purpose: Polycystic ovary syndrome (PCOS) is an endocrine-metabolic disorder that leads to lower natural reproductive potential and presents a challenge for assisted reproductive medicine because patients may exhibit immature oocyte retrieval and a higher risk of ovarian hyper stimulation syndrome during in vitro fertilization (IVF) treatment. This study aimed to identify potential lipid biomarkers for women with PCOS and a hyper response to controlled ovarian stimulation., Methods: Follicular fluid samples were collected from patients who underwent IVF, including normal responder women who became pregnant (control group, n = 11), women with PCOS and a hyper response to gonadotropins (PCOS group, n = 7) and women with only hyper response to gonadotropins (HR group, n = 7). A lipidomic analysis was performed by electrospray ionization mass spectrometry, and candidate biomarkers were analyzed by tandem mass spectrometry experiment., Results: The lipid profiles indicated particularities related to differences in phosphatidylcholine (PCOS and HR), phosphatidylserine, phosphatydilinositol and phosphatidylglycerol (control), sphingolipids (PCOS) and phosphatidylethanolamine (control and HR)., Conclusions: These findings contribute to the understanding of the molecular mechanisms associated with lipid metabolism in the PCOS-related hyper response, and strongly suggest that these lipids may be useful as biomarkers, leading to the development of more individualized treatment for pregnancy outcome.

The plasma levels of corticosteroids and sex steroids during pregnancy are key indicators of mammalian placental function and the onset of parturition. Steroid hormones are believed to be disturbed in pregnancies produced using assisted reproductive technologies (ARTs) due to placental dysfunction and the frequently observed lack of parturition signals. To elucidate the plasma steroid dynamics, a liquid chromatography-tandem mass spectrometry method was developed and used to determine the levels of corticosteroids (corticosterone, 11-deoxycortisol, and cortisol) and their direct precursors (progesterone and 17α-OH-progesterone) as well as sex steroids (androstenedione, estrone, estrone sulfate, testosterone, and 17β-estradiol) in bovine plasma. The levels of these 10 steroids in recipient cows carrying naturally conceived (control), in vitro fertilized (IVF), or cloned (somatic cell nuclear transfer) conceptuses were compared during late-term pregnancy (30 days before parturition), during near-term pregnancy (1 day before parturition), and on the day of parturition (day 0). Significant differences were observed among the corticosteroid levels: higher levels of corticosterone, 11-deoxycortisol, and cortisol were detected in cloned pregnancies at day 30; lower levels of corticosterone were observed in ART-derived pregnancies at days 1 and 0; and estrone and estradiol levels were higher in IVF pregnancies throughout the final development. These results suggested an upregulation of the P450C11 and P450C21 enzymes 30 days before parturition in somatic cell nuclear transfer pregnancies and an overactivation of the aromatase enzyme in IVF pregnancies. Taken together, the monitoring of multiple steroid hormones revealed that the pregnancies obtained using ART exhibited plasma steroid concentration dynamics compatible with the dysregulation of steroidogenic tissues.

Objective: Ovarian mucinous metastases commonly present as the first sign of the disease and are capable of simulating primary tumors. Our aim was to investigate the role of intratumoral lymphatic vascular density together with other surgical-pathological features in distinguishing primary from secondary mucinous ovarian tumors., Methods: A total of 124 cases of mucinous tumors in the ovary (63 primary and 61 metastatic) were compared according to their clinicopathological features and immunohistochemical profiles. The intratumoral lymphatic vascular density was quantified by counting the number of vessels stained by the D2-40 antibody., Results: Metastases occurred in older patients and were associated with a higher proportion of tumors smaller than 10.0 cm; bilaterality; extensive necrosis; extraovarian extension; increased expression of cytokeratin 20, CDX2, CA19.9 and MUC2; and decreased expression of cytokeratin 7, CA125 and MUC5AC. The lymphatic vascular density was increased among primary tumors. However, after multivariate analysis, the best predictors of a secondary tumor were a size of 10.0 cm or less, bilaterality and cytokeratin 7 negativity. Lack of MUC2 expression was an important factor excluding metastasis., Conclusions: The higher intratumoral lymphatic vascular density in primary tumors when compared with secondary lesions suggests differences in the microenvironment. However, considering the differential diagnosis, the best discriminator of a secondary tumor is the combination of tumor size, laterality and the pattern of expression of cytokeratin 7 and MUC2.

The antioxidant activity of Psychotria carthagenensis, P. leiocarpa, P. capillacea and P. deflexa (Rubiaceae) extracts were investigated, and the concentrations of total phenolics, flavonoids, condensed tannins and flavonols were determined. The chemical compositions of the extracts were investigated using the high performance liquid chromatography (HPLC/PAD) method. We used 1,1-diphenyl-1-picrylhydrazyl free radical (DPPH), β-Carotene bleaching and 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cations to determine antioxidant activity. The ability to scavenge radical was measured in these experiments by the discoloration of the solution. Concentrations of constituents were measured spectrophotometrically. P. carthagenensis and P. capillacea exhibited the highest antioxidant activity, in the DPPH test, β-carotene bleaching and ABTS system. The highest phenolic, flavonoid, condensed tannin and flavonol concentration was found in P. carthagenensis and P. capillacea extracts. HPLC-PDA analysis of P. carthagenensis and P. capillacea revealed hydroxycinnamic acid (p-coumaric acid). This is the first report on the antioxidant properties and constituent analysis of these Psychotria extracts.

The development of sensitive measurements to analyze individual cells is of relevance to elucidate specialized roles or metabolic functions of each cell under physiological and pathological conditions. Lipids play multiple and critical roles in cellular functions and the application of analytical methods in the lipidomics area is of increasing interest. In this work, in vitro maturation of porcine oocytes was studied. Two independent sources of chemical information (represented by mass spectra in the positive and negative ion modes) from single oocytes (immature oocytes, 24-h and 44-h in vitro matured oocytes) were acquired by using desorption electrospray ionization-mass spectrometry (DESI-MS). Low and mid-level data fusion strategies are presented with the aim of better exploring the large amount of chemical information contained in the two mass spectrometric lipid profiles. Data were explored by principal component analysis (PCA) within the two multi-block approaches to include information on free fatty acids, phospholipids, cholesterol-related molecules, di- and triacylglycerols. After data fusion, clearer differences among immature and in vitro matured porcine oocytes were observed, which provide novel information regarding lipid metabolism throughout oocyte maturation. In particular, changes in TAG composition, as well as increase in fatty acid metabolism and membrane complexity were evidenced during the in vitro maturation process. This information can assist the improvement of in vitro embryo production for porcine species., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Corynebacterium species (spp.) are among the most frequently isolated pathogens associated with subclinical mastitis in dairy cows. However, simple, fast, and reliable methods for the identification of species of the genus Corynebacterium are not currently available. This study aimed to evaluate the usefulness of matrix-assisted laser desorption ionization/mass spectrometry (MALDI-TOF MS) for identifying Corynebacterium spp. isolated from the mammary glands of dairy cows. Corynebacterium spp. were isolated from milk samples via microbiological culture (n=180) and were analyzed by MALDI-TOF MS and 16S rRNA gene sequencing. Using MALDI-TOF MS methodology, 161 Corynebacterium spp. isolates (89.4%) were correctly identified at the species level, whereas 12 isolates (6.7%) were identified at the genus level. Most isolates that were identified at the species level with 16 S rRNA gene sequencing were identified as Corynebacterium bovis (n=156; 86.7%) were also identified as C. bovis with MALDI-TOF MS. Five Corynebacterium spp. isolates (2.8%) were not correctly identified at the species level with MALDI-TOF MS and 2 isolates (1.1%) were considered unidentified because despite having MALDI-TOF MS scores >2, only the genus level was correctly identified. Therefore, MALDI-TOF MS could serve as an alternative method for species-level diagnoses of bovine intramammary infections caused by Corynebacterium spp., (Copyright © 2014 Elsevier B.V. All rights reserved.)

This research aimed to study the changes in lipid composition in cumulus cells using hyaluronidase according to the intracytoplasmic sperm injection protocol commonly used in human reproduction clinics. The lipid extraction was performed by the Blight-Dyer protocol and the lipid profiles were obtained by MALDI-TOF MS in positive and negative modes. The mass spectra data were processed with MassLynx and the statistical analysis was performed using MetaboAnalyst 2.0. Fifteen ions were selected for each mode as potential markers for differences between the groups. These ions were identified in the human metabolome database as phosphatidylserine with and without treatment, phosphatidylethanolamine in the after treatment group and phosphatidylinositol in the before treatment group, which are lipids that may be involved in cell apoptosis and signaling. We concluded that MALDI-TOF MS coupled with multivariate analysis can be utilized as a strategy to obtain and study the lipid profiles of cumulus cells and as a tool to study the metabolic state of cumulus cells.

Background: Increased potassium intake has been shown to lower blood pressure (BP) even in the presence of high sodium consumption however the role of dietary potassium on vascular function has received less attention. The aim of this study was to evaluate the relationship between habitual intake of sodium (Na) and potassium (K) and measures of arterial stiffness and wave reflection., Methods: Thirty-six young healthy adults (21 M, 15 F; 24 ± 0.6 yrs; systolic BP 117 ± 2; diastolic BP 63 ± 1 mmHg) recorded their dietary intake for 3 days and collected their urine for 24 hours on the 3rd day. Carotid-femoral pulse wave velocity (PWV) and the synthesis of a central aortic pressure waveform (by radial artery applanation tonometry and generalized transfer function) were performed. Aortic augmentation index (AI), an index of wave reflection, was calculated from the aortic pressure waveform., Results: Subjects consumed an average of 2244 kcals, 3763 mg Na, and 2876 mg of K. Average urinary K excretion was 67 ± 5.3 mmol/24 hr, Na excretion was 157 ± 11 mmol/24 hr and the average Na/K excretion ratio was 2.7 ± 0.2. An inverse relationship between AI and K excretion was found (r = -0.323; p < 0.05). A positive relationship between AI and the Na/K excretion ratio was seen (r = 0.318; p < 0.05) while no relationship was noted with Na excretion alone (r = 0.071; p > 0.05). Reflection magnitude, the ratio of reflected and forward waves, was significantly associated with the Na/K excretion ratio (r = 0.365; p <0.05) but not Na or K alone. PWV did not correlate with Na or the Na/K excretion ratio (p > 0.05) but showed an inverse relationship with K excretion (r = -0.308; p < 0.05)., Conclusions: These data suggest that lower potassium intakes are associated with greater wave reflection and stiffer arteries in young healthy adults.

This study identified possible lipid biomarkers in follicular fluid from women with poor ovarian response. These biomarkers indicate pathophysiological pathways and have potential diagnostic applications. An observational case-control study of young women undergoing ovarian stimulation for in-vitro fertilization was conducted. The participants were categorized into a poor ovarian response group and a normal ovarian response to stimulation group. All of the women underwent the same ovarian stimulation protocol, and follicular fluid was collected after ovarian aspiration. Analyses were performed using matrix-assisted laser desorption/ionization mass spectrometry. Principal component analysis and Volcano plots were used to describe follicular fluid classification models based on the lipid profiles. A total of 10 lipids were differentially expressed between the study and control groups. Of these lipid ions, three belonged to the phosphatidylcholine subclass and were present in higher concentrations in the control group. The other seven differential lipids were present in the study group and classified into four lipid subclasses: phosphatidylethanolamines, phosphatidylglycerols, phosphatidylinositols, and diacylglycerols. These distinctive lipids may be involved in hormonal responses and oocyte development processes and may be useful as biomarkers for therapeutic intervention in women with poor ovarian response.

Objective: To evaluate protein expression profile and to quantify proteins present in seminal plasma from men with spinal cord injury (SCI) and healthy men without SCI., Design: Experimental study., Setting: University hospital., Patient(s): Twelve SCI patients divided into two groups, six who underwent electroejaculation (EEJ) and six who underwent penile vibratory stimulation (PVS); and ten control subjects presenting normal sperm motility and concentration., Intervention(s): EEJ and PVS., Main Outcome Measure(s): The seminal plasma protein profile was analyzed by two proteomic strategies: data-independent label-free quantitative proteomics (MS(E)) and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE)., Result(s): A total of 638 different proteins were identified by MS(E) and 18 by 2D SDS-PAGE followed by tandem mass spectrometry. Interactome analysis showed key reproductive biologic processes-insemination, sperm and oocyte fusion, and acrosome reaction-related to all groups, as were triglyceride stimuli. Processes related to actin and muscle function and to iron oxidation, transportation, and homeostasis were found only in the EEJ and PVS groups; response to hydrogen peroxide and increased immune response was found only in the PVS group., Conclusion(s): This study was able to demonstrate differential protein expression among control, PVS, and EEJ groups; SCI is responsible for alterations in seminal plasma protein profile leading to a deviation from homeostasis; proteins reported in both PVS and EEJ groups correlate with the pathophysiology of SCI-related infertility., (Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Alteration of maternal lipid metabolism early in development has been shown to trigger obesity, insulin resistance, type 2 diabetes and cardiovascular diseases later in life in humans and animal models. Here, we set out to determine (i) lipid composition dynamics in single oocytes and preimplantation embryos by high mass resolution desorption electrospray ionization mass spectrometry (DESI-MS), using the bovine species as biological model, (ii) the metabolically most relevant lipid compounds by multivariate data analysis and (iii) lipid upstream metabolism by quantitative real-time PCR (qRT-PCR) analysis of several target genes (ACAT1, CPT 1b, FASN, SREBP1 and SCAP). Bovine oocytes and blastocysts were individually analyzed by DESI-MS in both positive and negative ion modes, without lipid extraction and under ambient conditions, and were profiled for free fatty acids (FFA), phospholipids (PL), cholesterol-related molecules, and triacylglycerols (TAG). Principal component analysis (PCA) and linear discriminant analysis (LDA), performed for the first time on DESI-MS fused data, allowed unequivocal discrimination between oocytes and blastocysts based on specific lipid profiles. This analytical approach resulted in broad and detailed lipid annotation of single oocytes and blastocysts. Results of DESI-MS and transcript regulation analysis demonstrate that blastocysts produced in vitro and their in vivo counterparts differed significantly in the homeostasis of cholesterol and FFA metabolism. These results should assist in the production of viable and healthy embryos by elucidating in vivo embryonic lipid metabolism.

In vitro-produced (IVP) bovine embryos are more sensitive to cryopreservation than their in vivo counterparts due to their higher lipid concentrations, whereas Bos indicus IVP embryos are even more sensitive than Bos taurus IVP embryos. To examine the effects of a lipolytic agent, before vitrification of Bos indicus IVP embryos, on embryo survival, viability, and pregnancy rates, two experiments were conducted. In experiment 1, Bos indicus (Nelore) embryos were produced from abattoir-derived ovaries and allocated into two groups. In the treatment group, 10 μM of forskolin was added to the in vitro culture medium on Day 5 and incubated for 48 hours. On Day 7 of culture, IVP-expanded blastocysts from both the control (n = 101) and treatment (n = 112) groups were vitrified with ethylene glycol and DMSO via the Cryotop procedure. Although there was no significant difference between the rates of blastocoel reexpansion and hatching of the embryos exposed to forskolin (87.5% and 70.5%, respectively) compared with the control embryos (79.2% and 63.3%, respectively), the numerically superior rates of the embryos exposed to forskolin led to another experiment. In experiment 2, blastocysts produced from the ovum pick up were exposed or not exposed to the lipolytic agent and vitrified as in experiment 1. Embryos treated with forskolin had higher pregnancy rates than the control group (48.8% vs. 18.5%). In view of these results, 1908 Bos indicus embryos were produced from ovum pick up, exposed to the lipolytic agent, and blastocysts were transferred to recipients, and the pregnancy rates of the embryos of various breeds were compared. The mean pregnancy rate obtained was 43.2%. All data were analyzed by chi-square or by binary logistic regression (P ≤ 0.05). In conclusion, treatment with forskolin before vitrification improved cryotolerance of Bos indicus IVP embryos, resulting in good post-transfer pregnancy rates., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Commercial cattle breeders produce their own herd offspring for the dairy and beef market using artificial insemination. The procedure involves sanitary risks associated with the collection and commercialization of the germplasm, and the in vitro production and transfer of the bovine embryos must be monitored by strict health surveillance. To avoid the spreading of infectious diseases, one must rely on using controlled and monitored germplasm, media, and reagents that are guaranteed free of pathogens. In this article, we investigated the use of a new mass spectrometric approach for fast and accurate identification of bacteria and fungi in bovine semen and in culture media employed in the embryo in vitro production process. The microorganisms isolated from samples obtained in a commercial bovine embryo IVP setting were identified in a few minutes by their conserved peptide/protein profile, obtained applying matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), matched against a commercial database. The successful microorganisms MS identification has been confirmed by DNA amplification and sequencing. Therefore, the MS technique seems to offer a powerful tool for rapid and accurate microorganism identification in semen and culture media samples., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In pre-implantation embryos, lipids play key roles in determining viability, cryopreservation and implantation properties, but often their analysis is analytically challenging because of the few picograms of analytes present in each of them. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) allows obtaining individual phospholipid profiles of these microscopic organisms. This technique is sensitive enough to enable analysis of individual intact embryos and monitoring the changes in membrane lipid composition in the early stages of development serving as screening method for studies of biology and biotechnologies of reproduction. This article introduces an improved, more comprehensive MALDI-MS lipid fingerprinting approach that considerably increases the lipid information obtained from a single embryo. Using bovine embryos as a biological model, we have also tested optimal sample storage and handling conditions before the MALDI-MS analysis. Improved information at the molecular level is provided by the use of a binary matrix that enables phosphatidylcholines, sphingomyelins, phosphatidylserines, phosphatidylinositols and phosphoethanolamines to be detected via MALDI(±)-MS in both the positive and negative ion modes. An optimal MALDI-MS protocol for lipidomic monitoring of a single intact embryo is therefore reported with potential applications in human and animal reproduction, cell development and stem cell research., (Copyright © 2013 John Wiley & Sons, Ltd.)