Polycystic ovary syndrome: clinical and laboratory variables related to new phenotypes using machine-learning models.

PURPOSE: Polycystic Ovary Syndrome (PCOS) is the most frequent endocrinopathy in women of reproductive age. Machine learning (ML) is the area of artificial intelligence with a focus on predictive computing algorithms. We aimed to define the most relevant clinical and laboratory variables related to PCOS diagnosis, and to stratify patients into different phenotypic groups (clusters) using ML algorithms. METHODS: Variables from a database comparing 72 patients with PCOS and 73 healthy women were included. The BorutaShap method, followed by the Random Forest algorithm, was applied to prediction and clustering of PCOS. RESULTS: Among the 58 variables investigated, the algorithm selected in decreasing order of importance: lipid accumulation product (LAP); abdominal circumference; thrombin activatable fibrinolysis inhibitor (TAFI) levels; body mass index (BMI); C-reactive protein (CRP), high-density lipoprotein cholesterol (HDL-c), follicle-stimulating hormone (FSH) and insulin levels; HOMA-IR value; age; prolactin, 17-OH progesterone and triglycerides levels; and family history of diabetes mellitus in first-degree relative as the variables associated to PCOS diagnosis. The combined use of these variables by the algorithm showed an accuracy of 86% and area under the ROC curve of 97%. Next, PCOS patients were gathered into two clusters in the first, the patients had higher BMI, abdominal circumference, LAP and HOMA-IR index, as well as CRP and insulin levels compared to the other cluster. CONCLUSION: The developed algorithm could be applied to select more important clinical and biochemical variables related to PCOS and to classify into phenotypically different clusters. These results could guide more personalized and effective approaches to the treatment of PCOS.

Low validity of predictive equations for calculating resting energy expenditure in overweight and obese women with polycystic ovary syndrome.

BACKGROUND: Predictive equations are the main clinical tools for determining resting energy expenditure (REE). However, their adequate use in overweight and obese individuals is unclear. Thus, we investigated the best predictive equations for estimating REE in overweight and obese women with polycystic ovary syndrome (PCOS). METHODS: Eleven analyses were performed with prediction equations (pREE) based on anthropometric parameters in 30 overweight or obese women with PCOS without other chronic diseases. The measured REE (mREE) was calculated by indirect calorimetry. The validity of the equations was investigated by comparison, accuracy and agreement tests between pREE and mREE at both the individual and group level. RESULTS: Four analyses were similar to those of mREE, and smallest mean differences were observed for the World Health Organization/Food and Agriculture Organization of the United Nations/United Nations University (WHO/FAO/UNU) considering weight (W) [0.07 (1.13) MJ (16 [270] kcal)]. Individual accuracy was greater than 50% for Harris and Benedict, Müller and Lazzer equations. The percentage of REE underestimation ranged between 16.7% and 73.3%, whereas higher rates of overestimation were observed in the De Luis (66.7%) and Ireton-Jones (43.3%) equations. Mean bias at the group level was lowest in the WHO/FAO/UNU W and WHO/FAO/UNU considering weight and height (WH), Müller and Lazzer equations (-2.8 to 0.5). The WHO/FAO/UNU W and WHO/FAO/UNU WH formulas were optimal in individual agreement (33.3%). CONCLUSIONS: FAO/WHO/UNU W equations may estimate the REE in overweight and obese women with PCOS. However, the low individual accuracy and agreement in relation to mREE suggest caution regarding when to use the formula to perform an individual nutritional plan.

Non-alcoholic fatty liver disease in women with polycystic ovary syndrome: systematic review and meta-analysis.

PURPOSE: Non-alcoholic fatty liver disease (NAFLD) is an insidious pathologic condition that can manifest from simple steatosis to steatohepatitis (NASH) with potential progression to cirrhosis. Like the polycystic ovary syndrome (PCOS), NAFLD is associated with obesity, diabetes mellitus, insulin resistance and metabolic syndrome. PCOS women have an increased risk of NAFLD, but it is debatable which features of PCOS, either specific (androgen excess) or unspecific (metabolic derangements) affect the NAFLD risk. METHODS: We performed a systematic review and meta-analysis of studies that addressed the association of PCOS and NAFLD. We selected 17 studies published between 2007 and 2017 that included 2734 PCOS patients and 2561 controls of similar age and body mass index (BMI). RESULTS: PCOS patients have increased prevalence of NAFLD (odds ratio 2.54, 95% confidence interval 2.19-2.95). PCOS women with hyperandrogenism (classic phenotype) have a higher prevalence of NAFLD compared to women with PCOS without hyperandrogenism, even after correction for confounding variables. Among women with PCOS, those with NAFLD have higher serum total testosterone (mean difference 0.40 nmol/L, 95% CI 0.29-0.50 nmol/L) and free androgen index (mean difference 4.46, 95% CI 3.53-5.39) than those without NAFLD. The studies that used multivariate analysis controlling for age, BMI, triglycerides, and insulin resistance index confirmed that serum androgens are independent predictors of NAFLD in women with PCOS. CONCLUSION: The prevalence of NAFLD is increased in women with PCOS and the presence of NAFLD is associated with high serum androgen levels, in addition to obesity and insulin resistance.

Microparticles: Inflammatory and haemostatic biomarkers in Polycystic Ovary Syndrome.

Polycystic Ovary Syndrome (PCOS) is associated with a chronic low-grade inflammation and predisposition to hemostatic and atherosclerotic complications. This case-control study evaluated the microparticles (MPs) profile in patients with the PCOS and related these MPs to clinical and biochemical parameters. MPs derived from platelets (PMPs), leuckocytes (LMPs) and endothelial cells (EMPs) were evaluated, as well as MPs expressing tissue factor (TFMPs), by flow cytometry, comparing women with PCOS (n = 50) and a healthy control group (n = 50). PCOS women presented increased total MPs, PMPs, LMPs and EMPs levels when compared to control group (all p < 0.05). TFMPs was similar between the groups (p = 0.379). In conclusion, these MPs populations could be useful biomarkers for association with thrombosis and cardiovascular disease in PCOS women.

Molecular typing of Salmonella from Sergipe, Northeastern Brazil, showing the epidemiological relationship between poultry and human infection.

Randomly amplified polymorphic DNA (RAPD) has been widely used for epidemiological and phylogenetic purposes ow-ing to its rapidity and efficiency. The aim of this study was to perform genome typing of Salmonella samples isolated from different sources by RAPD profiling. Thirty-three Salmonella samples from the bacterial collection of the Laboratório de Virologia Comparada, Departamento de Morfologia, Universidade Federal de Sergipe, Brazil, and two standard samples were used. RAPD profiling was conducted using six primers of the Ready-To-Go RAPD system. The amplified products were electro-phoresed on 5% polyacrylamide gel and silver-stained. RAPD analysis resulted in reproducible and stable banding patterns and showed high genetic diversity among the isolated strains. The Primer P1-generated dendrogram showed an epidemiologic relationship between the human and poultry isolated samples, highlighting the usefulness of RAPD for molecular typing and epidemiological studies.

Estrogen receptor αlpha gene (ESR1) PvuII and XbaI polymorphisms are associated to metabolic and proinflammatory factors in polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a complex endocrine disorder that involves multiple factors. Although the etiology of PCOS is unknown, there is an involvement of sex steroid hormones in the pathophysiology of this syndrome. Therefore, polymorphisms in genes involved in the action of estrogen may contribute to a woman's susceptibility to PCOS. AIM: This study aimed to evaluate the association between the polymorphisms PvuII and XbaI in the estrogen receptor alpha (ESR1) gene and the occurrence of PCOS. The study also aimed to assess the influence of these polymorphisms on the metabolic and inflammatory profiles of women with PCOS. MATERIAL AND METHODS: This case-control study included 99 women with PCOS, diagnosed according to the Rotterdam criteria, and 104 age-matched healthy women. The polymorphisms were evaluated using polymerase chain reaction-restriction fragment length polymorphism. RESULTS: No association between the ESR1 gene polymorphisms and the presence of PCOS was observed. However, we found associations between the PvuII polymorphism and C-reactive protein levels, testosterone levels, family history of diabetes, and waist circumference. The XbaI polymorphism was associated with fasting glucose and a family history of hypertension. CONCLUSION: These polymorphisms are not associated with PCOS development, but they are involved in the phenotype of complications of the syndrome. Therefore, prior knowledge of these genomic variants might contribute to taking preventive measures that could delay the metabolic and reproductive complications commonly seen in women with PCOS.

Poor quality diet is associated with overweight status and obesity in patients with polycystic ovary syndrome.

BACKGROUND: The high rates of overweight status observed in women with polycystic ovary syndrome (PCOS) may reflect dietary intake, and so it is important to investigate diet quality and its relationship with the rates of overweight status and obesity among these patients. METHODS: A cross-sectional study was conducted in which 100 women with PCOS (Rotterdam criteria) were evaluated considering anthropometric and dietary data. The anthropometric evaluation included the measurement of weight, height, body mass index (BMI), waist circumference (WC), hip circumference and waist-hip ratio. Food intake data were collected from two 24-h dietary recalls to assess dietary patterns using the Brazilian Healthy Eating Index - Revised (BHEI-R). RESULTS: The anthropometric analysis indicated a high prevalence of overweight status, obesity and increased visceral fat (30.0%, 60.0% and 90.0%, respectively). The mean BHEI-R score was 56.1 ± 12.0 points (range 34.5-77.5 points). Diet quality was negatively correlated with obesity, which was evaluated by BMI (r = -0.248; P = 0.013) and WC (r = -0.278; P = 0.005). CONCLUSIONS: Dietary interventions focused on improvement of diet quality should be targeted to treat patients with PCOS because obesity in these women is associated with worsening endocrine, metabolic and reproductive functions.

Correlation between plasminogen activator inhibitor-1 (PAI-1) promoter 4G/5G polymorphism and metabolic/proinflammatory factors in polycystic ovary syndrome.

Polycystic Ovary Syndrome (PCOS) is the most common cause of subfertility associated to metabolic disorders. The aim of this study was to correlate metabolic and proinflammatory factors in women with PCOS. The frequency of Plasminogen Activator Inhibitor-1 (PAI-1) promoter 4 G/5 G polymorphism was also compared to healthy controls. We evaluated 79 PCOS and 79 healthy women. PAI-1 levels are positively correlated with proinflammatory factors in PCOS group. 4 G allele in PAI-1 gene was more frequent in PCOS and the 4G/4 G genotype was associated with increased PAI-1 levels. A correlation between insulin resistance and proinflammatory and overweight was also observed. C-reactive protein, serum levels of vascular cell adhesion molecule-1 (sVCAM-1), Lipid Accumulation Product (LAP) and vitamin D are good tools to evaluated factors associated to cardiovascular risk in women with PCOS.

Serological responses in sheep injected with plasmids encoding bovine herpesvirus 1 (BHV-1) gD glycoprotein / Resposta sorológica de ovinos inoculados com plasmídeos codificando a glicoproteína D do herpesvírus bovino 1 (BHV-1)

A genetic vaccine consisting of the bovine herpesvirus-1.2a (BHV-1.2a) glycoprotein D (gD) gene under the control of the cytomegalovirus immediate-early promoter/enhancer was generated and administered to sheep intramuscularly in the neck. All animals developed serum antibodies which recognized the homologous antigen (BHV-1.2a strain BH-83) and also exhibited cross-reactivity against the heterologous antigen (BHV-5 strain EVI-190). Three intramuscularly injections were given but serological responses were not improved after the second inoculation. Specific antibodies were detected against BHV-1.2a until at least 12 months after the first inoculation. However, the capacity to induce antibodies against BHV-5 was lower and of shorter duration than to BHV-1.2a.(AU)

Uma vacina geneticamente preparada com o gene da glicoproteína D do herpesvirus bovino-1.2a (BHV-1.2a) controlado com o promotor/potenciador imediatamente precoce de citomegalovírus foi administrada no pescoço de ovinos. Todos os animais produziram anticorpos séricos que reconheceram, em ELISA, o antígeno homólogo (BHV-1.2a, amostra BH-83) e também mostraram reatividade cruzada contra um antígeno heterólogo (BHV-5 amostra EVI-190). Foram aplicadas três inoculações, mas não houve melhora de resposta com a terceira inoculação. Resposta específica contra BHV-1.2a foi detectada pelo menos até 12 meses após a primeira inoculação, entretanto, a resposta sorológica contra o BHV-5 foi de menor intensidade e duração do que aquela contra o BHV-1.2a.(AU)

Serological responses in sheep injected with plasmids encoding bovine herpesvirus 1 (BHV-1) gD glycoprotein

A genetic vaccine consisting of the bovine herpesvirus-1.2a (BHV-1.2a) glycoprotein D (gD) gene under the control of the cytomegalovirus immediate-early promoter/enhancer was generated and administered to sheep intramuscularly in the neck. All animals developed serum antibodies which recognized the homologous antigen (BHV-1.2a strain BH-83) and also exhibited cross-reactivity against the heterologous antigen (BHV-5 strain EVI-190). Three intramuscularly injections were given but serological responses were not improved after the second inoculation. Specific antibodies were detected against BHV-1.2a until at least 12 months after the first inoculation. However, the capacity to induce antibodies against BHV-5 was lower and of shorter duration than to BHV-1.2a.

Caracterizaçäo parcial de uma amostra brasileira do vírus da doença de Aujeszky isolada / Partial characterization of a Brazilian strain of Aujeszky's disease virus recovered from a pig with subclinical infection

Uma amostra brasileira do vírus da doença de Aujeszky, isolada de um leitäo, foi estudada quanto à virulência para suínos e seu genoma analisado no que se refere ao padräo de fragmentaçäo pela enzima de restriçäo Bam HI. Leitöes com 50 dias de idade, sorologicamente negativos, expostos a essa amostra, näo mostraram sinais de doença e o vírus foi detectado na regiäo orofaríngeana durante pelo menos três dias após exposiçäo. Com base no padräo de migraçäo dos fragmentos de restriçäo, o isolamento foi classificado como pertencente ao grupo I da classificaçäo proposta por Herrmann. Infecçäo latente foi detectada por PCR em todos os suínos. Algumas variaçöes foram identificadas no mapa físico de Bam HI da amostra ASB Piau quando se comparou com o mapa obtido da amostra virulenta brasileira LA03l

Detecçäo do vírus da hepatite murina utilizando-se a reaçäo em cadeia pela polimerase / Detection of mouse hepatitis virus in mouse colonies using the nested polymerase chain reaction

Desenvolveu-se a técnica de transcriçäo reversa - reaçäo em cadeia pela polimerase para detectar o vírus da hepatite do camundongo em tecido hepático. Para se eliminar possíveis falhas na reaçäo de amplificaçäo RT-PCR usou-se um segundo ciclo de amplificaçäo com primers internos para confirmar a especificidade e aumentar a sensibilidade do teste. Após a optimizaçäo do protocolo, descreve-se a detecçäo da amplificaçäo específica da seqüência-alvo em camundongos de 18 das 20 colônias examinadas

Polymerase chain reaction amplification of genomic fragments of bovine herpesvirus-1

Especial conditions were developed for the amplification of five DNA segments from US region of BHV-1 by polymerase chain reaction. In order to eliminate most nonspecific products it was found that addition of three cosolvents DMSO, glycerol and NP 40 was a simple method for increasing the specificity of amplification.

Detection of mouse hepatitis virus in mouse colonies using the nested polymerase chain reaction

A reverse transcriptase polymerase chain reaction (RT-PCR) to detect mouse hepatitis virus (MHV) in hepatic tissue was developed. To circumvent possible failures in RT-PCR amplifications, a second round of PCR with internal primers was used to confirm the specificity and increase the sensitivity of the test. Using this method specific amplification of MHV sequences was observed in 18 out of 20 mouse colonies examined.

Desenvolveu-se a técnica de transcrição reversa - reação em cadeia pela polimerase para detectar o vírus da hepatite do camundongo em tecido hepático. Para se eliminar possíveis falhas na reação de amplificação RT-PCR usou-se um segundo ciclo de amplificação com primers internos para confirmar a especificidade e aumentar a sensibilidade do teste. Após a optimização do protocolo, descreve-se a detecção da amplificação específica da seqüência-alvo em camundongos de 18 das 20 colônias examinadas.

Partial characterization of a Brazilian strain of Aujeszkys disease virus recovered from a pig with subclinical infection

One Brazilian strain of Aujeszkys disease virus isolated from a piglet in which the disease had not been observed was studied as for its virulence in pigs. The genome of the virus was molecularly analysed as for their restriction endonuclease cleavage pattern. Fifty-day-old non-immune weanlings exposed to this strain showed no disease although the virus was present in their oropharyngeal area for at least three days. All animals developed moderate titers of neutralizing antibody. Based on number of bands and migration rate of restriction fragments the isolate was classified into Herrmanns type I group. Latent infection was detected in all pigs by PCR. Some variations were detected in the cleavage pattern of the strain ASB Piau when compared to LA031 virulent Brazilian strain, that could be related to differences in the virulence.

Uma amostra brasileira do vírus da doença de Aujeszky, isolada de um leitão, foi estudada quanto à virulência para suínos e seu genoma analisado no que se refere ao padrão de fragmentação pela enzima de restrição Bam HI. Leitões com 50 dias de idade, sorologicamente negativos, expostos a essa amostra, não mostraram sinais de doença e o vírus foi detectado na região orofaríngeana durante pelo menos três dias após exposição. Com base no padrão de migração dos fragmentos de restrição, o isolamento foi classificado como pertencente ao grupo I da classificação proposta por Herrmann. Infecção latente foi detectada por PCR em todos os suínos. Algumas variações foram identificadas no mapa físico de Bam HI da amostra ASB Piau quando se comparou com o mapa obtido da amostra virulenta brasileira LA031.

Desenvolvimento de uma sonda de DNA para a detecçäo do vírus da doença de Aujeszky / Development of DNA probe for detection of Aujeszky's disease virus

Uma sonda radioativa e uma outra biotinilada foram produzidas para detecçäo do vírus da doença de Aujeszky (VDA). O fragmento de Bam H I 7 do DNA genômico do VDA foi marcado por "nick translation" empregando P32dCTP e um fragmento de 196pb, resultante de uma amplificaçäo pela reaçäo em cadeia pela polimerase marcado com biotina 7-dATP. Essas sondas, de uma maneira rápida e específica, prestaram-se para a detecçäo da infecçäo aguda pelo VDA. Entretanto, näo se mostraram sensíveis o suficiente para detectar seqüências genômicas de VDA no gânglio trigêmio de suínos e camundongos com infecçäo latente

Detecçäo de amostras brasileiras do herpesvirus bovino 1 (BHV-1) pela reaçäo em cadeia pela polimerase / Detection of different Brazilian strains of the bovine herpesvirus-1 (BHV-1) by polymerase chain reaction

A técnica da reaçäo em cadeia pela DNA polimerase (PCR) foi usada para a amplificaçäo rápida de um fragmento de 456bp da regiäo única curta (Us) do genoma do BHV-1. Iniciadores de 18pb do gene da ORF1 foram usados para a amplificaçäo das amostras-padräo e brasileiras. Uma amplificaçäo clássica näo foi bem sucedida. A amplificaçäo foi obtida quando se escolheu uma regiäo com baixa concentraçäo de GC no DNA do BHV-1 e através da desnaturaçäo térmica (95§C para 5min) seguida de ciclos térmicos (94§C por 1min e 30seg; 52§C por 1min; 72§C por 1min e 30 seg; e ainda por 35 ciclos de 94§C por 1min; 52§C por 1min; 72§C por 1min e 30 seg; usando um tempo de extensäo final de 72§C por 5min). A clivagem com Pst I confirmou a especificidade do fragmento da ORF 1 do BHV-1. A amplificaçäo do fragmento em todas as amostras testadas sugere que a regiäo é fortemente conservada no genoma do BHV-1. Este PCR poderá detectar rapidamente amostras clínicas, sendo sensível e específico para diagnosticar infecçöes pelo BHV-1

Main features of DNA-based immunization vectors.

DNA-based immunization has initiated a new era of vaccine research. One of the main goals of gene vaccine development is the control of the levels of expression in vivo for efficient immunization. Modifying the vector to modulate expression or immunogenicity is of critical importance for the improvement of DNA vaccines. The most frequently used vectors for genetic immunization are plasmids. In this article, we review some of the main elements relevant to their design such as strong promoter/enhancer region, introns, genes encoding antigens of interest from the pathogen (how to choose and modify them), polyadenylation termination sequence, origin of replication for plasmid production in Escherichia coli, antibiotic resistance gene as selectable marker, convenient cloning sites, and the presence of immunostimulatory sequences (ISS) that can be added to the plasmid to enhance adjuvanticity and to activate the immune system. In this review, the specific modifications that can increase overall expression as well as the potential of DNA-based vaccination are also discussed.

Main features of DNA-based immunization vectors

DNA-based immunization has initiated a new era of vaccine research. One of the main goals of gene vaccine development is the control of the levels of expression in vivo for efficient immunization. Modifying the vector to modulate expression or immunogenicity is of critical importance for the improvement of DNA vaccines. The most frequently used vectors for genetic immunization are plasmids. In this article, we review some of the main elements relevant to their design such as strong promoter/enhancer region, introns, genes encoding antigens of interest from the pathogen (how to choose and modify them), polyadenylation termination sequence, origin of replication for plasmid production in Escherichia coli, antibiotic resistance gene as selectable marker, convenient cloning sites, and the presence of immunostimulatory sequences (ISS) that can be added to the plasmid to enhance adjuvanticity and to activate the immune system. In this review, the specific modifications that can increase overall expression as well as the potential of DNA-based vaccination are also discussed

Development of DNA probe for detection of Aujeszky's disease virus

A DNA hybridization dot-blot assay using a radioactive and a non-radioactive probe has been developed for the detection of Aujeszky's disease virus (ADV). The Bam H I 7 fragment of ADV genomic DNA was labeled by nick translation using 32P-dCTP and the 196bp polymerase chain reaction (PCR) gG glycoprotein gene amplified fragment was also labeled by nick translation but using biotin d-7-dATP. This technique provides a fast and effective means of detecting acute cases of ADV infection but it was unable to detect ADV nucleic acid sequences in trigeminal nerve ganglia of latent infected pigs and mice.

Uma sonda radioativa e uma outra biotinilada foram produzidas para detecção do vírus da doença de Aujeszky (VDA). O fragmento de Bam H I 7 do DNA genômico do VDA foi marcado por "nick translation" empregando P32dCTP e um fragmento de 196pb, resultante de uma amplificação pela reação em cadeia pela polimerase marcado com biotina 7-dATP. Essas sondas, de uma maneira rápida e específica, prestaram-se para a detecção da infecção aguda pelo VDA. Entretanto, não se mostraram sensíveis o suficiente para detectar seqüências genômicas de VDA no gânglio trigêmio de suínos e camundongos com infecção latente.