Sirtuins in tumorigenesis

Sirtuins (SIRT) are group of enzymes that require nicotinamide adenine dinucleotide (NAD+) to catalyze their reactions. These chemical compounds have mono (ADP-ribosyl) transferase or deacetylases activities, and they can be found in nearly all species. The mammalian sirtuin family is described by seven proteins, namely. Every group of sirtuins can be found in the different regions of the cells; SIRT1 is predominantly nuclear, SIRT2 is located mainly in the cytoplasm (but it can shuttle between the nucleus and the cytoplasm), SIRT3, SIRT4, and SIRT5 are mitochondrial proteins, (SIRT3 can move from the nucleus to mitochondria during cellular stress), SIRT6 and SIRT7 are nuclear sirtuins. Sirtuins have a lot of functions in different physiological processes such as gene repression, metabolic control, apoptosis and cell survival, DNA repair, development, inflammation, neuroprotection, and healthy aging. Because of so many roles in physiological processes there is a huge interest not just in their functions but also in the different compounds which can modify their functions. In this article we will focus on the role of sirtuins in tumorigenesis

Redefinition of gestational diabetes mellitus: implications for laboratory practice in Croatia

An increasing prevalence of gestational diabetes has become a very challenging task in prenatal care worldwide. International Association of Diabetes and Pregnancy Study Groups (IADPSG) has recently issued recommendations on the diagnosis and classification of hyperglycaemia in pregnancy. These recommendations, the first to provide harmonised, evidence-based criteria for the diagnosis and classification of diabetes in pregnancy, are currently being discussed and accepted worldwide by the relevant authorities. As the acceptance of the proposed criteria has major implications for both clinical and laboratory settings, a concerted action towards necessary changes in practice has to be carefully planned and adjusted to national health-care specificities. IADPSG criteria have been strongly advocated by the Croatian Perinatology Society, resulting in a new strategy for the detection and diagnosis of hyperglycaemic disorders in pregnancy. To address the respective laboratory requirements, in April 2012, the Croatian Chamber of Medical Biochemists appointed a Working Group to provide a standardised procedure for the diagnosis of gestational diabetes, applicable to all laboratories involved in prenatal care, in both primary and specialised health-care facilities. In this paper we discuss key laboratory-related issues regarding succesful implementation of the IADPSG criteria in Croatia

COMBINED ULTRASOUND-BIOCHEMICAL SCREENING OF FETAL TRISOMIA IN THE FIRST TRIMESTER AND DOUBLE BIOCHEMICAL SCREENING IN THE SECOND TRIMESTER AT NONRISK PREGNANCIES

Cilj rada: Usporediti uspješnost primjene probirnih testova aneuploidija u prvom i drugom tromjesečju trudnoće. Ispitanice i metode: Istraživanu skupinu činilo je 374 trudnica s kombiniranim ultrazvučno-biokemijskim testom u prvom, odnosno dvostrukim biokemijskim testom u drugom tromjesečju. Sve su trudnoće bile jednoplodne i urednog ishoda. U probiru prvog tromjesečja korišteni su biokemijski biljezi u serumu trudnica: plazmatski protein pridružen trudnoći (PAPP-A) i slobodna B-podjedinica humanog korionskog gonadotropina (slobodni B-hCG), uz ultrazvukom izmjerenu debljinu nuhalnog nabora (NT) i dužinu tjeme-trtica (CRL) u ploda. U dvostrukom biokemijskom probiru drugog tromjesečja korišteni su biokemijski biljezi: alfa-fetoprotein (AFP) i slobodni B-hCG, a gestacija je procijenjena prema ultrazvučnoj biometriji. Rezultati: Povećani ultrazvučno-biokemijski rizik trisomije 21 u 1. tromjesečju trudnoće ustanovili smo u 30 trudnica (8.0%). Od ukupnog broja, 70 ispitanica (18.7%) imalo je povećani rizik obzirom na biokemijske biljege u ¬prvom, odnosno njih 56 (15.0%) obzirom na biokemijske biljege u drugom tromjesečju trudnoće. Izvršeno je ukupno 30 postupaka amniocenteze. Od toga je u 19 trudnica (63.3%) indikacija postavljena na osnovi kombiniranog probirnog testa. Nadalje, amniocenteza je izvršena u 11 trudnica (28.2%) zbog povećanog rizika u dvostrukom biokemijskom probiru drugog tromjesečja. Udjel lažno-pozitivnih razultata u kombiniranom probiru prvog tromjesečja bio je statistički značajno niži, nego u biokemijskom probiru drugog tromjesečja (B2=12.02, p=0.0005). Ustanovili smo značajnu pozi¬tivnu povezanost log10 MoM slobodnog B-hCG između prvog i drugog tromjesečja (r2=0.403, p<0.0001). Nismo ustanovili značajnu povezanost između ostalih biokemijskih biljega u prvom, odnosno, drugom tromjesečju. Zaključak: Rezultati našeg istraživanja su pokazali značajno veću specifičnost ranog kombiniranog probirnog testa aneuploidija u odnosu na biokemijski probirni test u prvom, kao i u drugom tromjesečju. Mogućnosti izbora pojedinih probirnih testova trebale bi biti usklađene sa stavovima i potrebama samih trudnica, kao i sa smjernicama koje su preporučile nadležne institucije za fetalnu medicinuObjective: To evaluate the performance of screening tests for aneuploidy in the first and second trimesters of pregnancy in Croatian pregnant women. Study Design and Methods: Study population comprised 374 pregnant women who underwent the combined ultrasound-biochemical in the first and double-test in the second trimester of pregnancy, respectively. All were singleton pregnancies with normal outcomes. The first-trimester screening was performed combining serum markers, pregnancy associated plasma protein-A (PAPP-A) and free B-subunit of human chorionic gonado¬tropin (free B-hCG) with fetal nuchal translucency thickness (NT) and crown-rump length (CRL), measured by ultrasound. For the second-trimester screening, maternal serum alpha-fetoprotein and free B-hCG were used as biochemical markers in relation to fetal biometry diagnosed by ultrasound. Results: In 30 pregnant women (8.0%) elevated risk for trisomy 21 was found after combined ultrasound-biochemical screening. Out of total, 70 pregnant women (18.7%) were classified ’at risk’ on the basis of biochemical markers in the first and 56 (15.0%) in the second trimester. In 30 pregnant women amniocentesis was performed. In 19 cases (63.3%) the indication was elevated risk in the first-trimester combined test. In 11 women (28.2%) amniocentesis is recommended because of the elevated second-trimester biochemical risk. The proportion of false-positive results in combined first-trimester screening test was significantly lower than with second-trimester biochemical markers (B2=12.02, p=0.0005). We found the significant positive relationship between log10 MoM F B-hCG in the first and second trimester (r2=0.403, p<0.0001). There was no significant relationship between PAPP-A and second-trimester biochemical markers. Conclusion: Results of this preliminary study confirmed better specificity of the combined first-trimester screening in relation to biochemical screening in the second trimester of pregnancy. The decision and choice of the most appropriate screening test should consider woman’s personal attitude and preferences, as well as follow the guidelines recommended by the competent associations for the fetal medicine

COMBINED ULTRASOUND AND BIOCHEMICAL SCREENING FOR FETAL ANEUPLOIDY AT 10 – 14 WEEKS OF PREGNANCY: FIRST RESULTS OF TEST PERFORMANCE IN CROATIA

Sažetak. Cilj rada. Retrospektivna studija nakon primjene kombiniranog ultrazvučno-biokemijskog testa probira trisomija u prvom tromjesečju trudnoće. Metode. Od veljače 2006. do srpnja 2008. godine probir je učinjen u 1112 trudnica između 10. i 14. tjedna trudnoće. Individualni rizik trisomije 21, 18 i 13 izračunavali smo kombinacijom dobnog rizika trudnice, ultrazvučnih biljega u ploda (debljina nuhalnog nabora – NT, udaljenosti tjeme-trtica – CRL) te biokemijskih biljega u serumu trudnice (slobodni -hCG i PAPP-A), pomoću licenciranog računalnog programa (Typolog). Koncentracije biokemijskih biljega smo određivali imunometrijskom kemiluminiscentnom metodom (IMMULITE). Biokemijske biljege, kao i NT u odnosu na CRL, izrazili smo u obliku višekratnika MoM, u odnosu na dnevne regresijske medijane za odgovarajuću gestaciju u neugroženim trudnoćama. Rezultate smo obradili nakon dovršenih svih ispitanih trudnoća. Ukupno su 62 testirane trudnice imale povećani kombinirani rizik trisomije 21, od kojih je 10 trudnica imalo i povećani rizik trisomije 18/13. Četiri trisomije 21 i jedna trisomija 18 otkrivene su prenatalnom dijagnozom; stopa detekcije bila je 100% (5/5). U trudnica s povećanim rizikom u probiru učinjeno je 7 biopsija koriona i 38 ranih amniocenteza. Udio lažno-pozitivnih rezultata bio je 5.1%. Zaključak. Prvi rezultati provođenja kombiniranog probirnog testa u Hrvatskoj potvrdili su visoku osjetljivost i veću specifičnost, u poredbi s biokemijskim probirnim testom u drugom tromjesečju trudnoće.Objective. Retrospective study of the results of the first-trimester combined screening for fetal trisomies with ultrasound and biochemical markers. Methods. In the period from February 2006 to July 2008, a total of 1112 pregnant-women underwent screening between the 10th and 14th gestational week. Individual risk for trisomies 21, 18 and 13, combining maternal age, ultrasonography (nuchal translucency, crown-rump length) and serum biochemical analytes (free -hCG, PAPP-A) was computed by means of licensed Typolog software. Concentrations of biochemical markers were determined by chemiluminiscent immunometric assay (IMMULITE). Both biochemical markers, as well as NT, were expressed as Multiples of the Median (MoM), based on the regressed medians of the corresponding gestational age in unaffected pregnancies. Results. All studied pregnancies were followed up to term. A total of 62 pregnant women were categorized as high-risk for trisomy 21, and 10 of them had also an elevated risk for trisomies 18/13, respectively. Four trisomies 21 and one trisomy 18 were detected through combined test and confirmed with prenatal diagnostic procedure. Detection rate was 100%. In those with high risk, 7 chorionic villi sampling and 38 amniocenteses were performed. False-positive rate was 5.1%. Conclusion. The results of the first-trimester screening in Croatia confirmed high sensitivity and better specificity of the combined ultrasonic and biochemical markers, in relation with the second-trimester biochemical screening test

COMBINED ULTRASOUND AND BIOCHEMICAL SCREENING FOR FETAL ANEUPLOIDY AT 10 – 14 WEEKS OF PREGNANCY: FIRST RESULTS OF TEST PERFORMANCE IN CROATIA

Sažetak. Cilj rada. Retrospektivna studija nakon primjene kombiniranog ultrazvučno-biokemijskog testa probira trisomija u prvom tromjesečju trudnoće. Metode. Od veljače 2006. do srpnja 2008. godine probir je učinjen u 1112 trudnica između 10. i 14. tjedna trudnoće. Individualni rizik trisomije 21, 18 i 13 izračunavali smo kombinacijom dobnog rizika trudnice, ultrazvučnih biljega u ploda (debljina nuhalnog nabora – NT, udaljenosti tjeme-trtica – CRL) te biokemijskih biljega u serumu trudnice (slobodni -hCG i PAPP-A), pomoću licenciranog računalnog programa (Typolog). Koncentracije biokemijskih biljega smo određivali imunometrijskom kemiluminiscentnom metodom (IMMULITE). Biokemijske biljege, kao i NT u odnosu na CRL, izrazili smo u obliku višekratnika MoM, u odnosu na dnevne regresijske medijane za odgovarajuću gestaciju u neugroženim trudnoćama. Rezultate smo obradili nakon dovršenih svih ispitanih trudnoća. Ukupno su 62 testirane trudnice imale povećani kombinirani rizik trisomije 21, od kojih je 10 trudnica imalo i povećani rizik trisomije 18/13. Četiri trisomije 21 i jedna trisomija 18 otkrivene su prenatalnom dijagnozom; stopa detekcije bila je 100% (5/5). U trudnica s povećanim rizikom u probiru učinjeno je 7 biopsija koriona i 38 ranih amniocenteza. Udio lažno-pozitivnih rezultata bio je 5.1%. Zaključak. Prvi rezultati provođenja kombiniranog probirnog testa u Hrvatskoj potvrdili su visoku osjetljivost i veću specifičnost, u poredbi s biokemijskim probirnim testom u drugom tromjesečju trudnoće.Objective. Retrospective study of the results of the first-trimester combined screening for fetal trisomies with ultrasound and biochemical markers. Methods. In the period from February 2006 to July 2008, a total of 1112 pregnant-women underwent screening between the 10th and 14th gestational week. Individual risk for trisomies 21, 18 and 13, combining maternal age, ultrasonography (nuchal translucency, crown-rump length) and serum biochemical analytes (free -hCG, PAPP-A) was computed by means of licensed Typolog software. Concentrations of biochemical markers were determined by chemiluminiscent immunometric assay (IMMULITE). Both biochemical markers, as well as NT, were expressed as Multiples of the Median (MoM), based on the regressed medians of the corresponding gestational age in unaffected pregnancies. Results. All studied pregnancies were followed up to term. A total of 62 pregnant women were categorized as high-risk for trisomy 21, and 10 of them had also an elevated risk for trisomies 18/13, respectively. Four trisomies 21 and one trisomy 18 were detected through combined test and confirmed with prenatal diagnostic procedure. Detection rate was 100%. In those with high risk, 7 chorionic villi sampling and 38 amniocenteses were performed. False-positive rate was 5.1%. Conclusion. The results of the first-trimester screening in Croatia confirmed high sensitivity and better specificity of the combined ultrasonic and biochemical markers, in relation with the second-trimester biochemical screening test

Clinical and biochemical characteristics of polycystic ovary syndrome in Croatian population [Kliničke i biokemijske karakteristike bolesnica sa sindromom policističnih jajnika u Hrvatskoj populaciji]

Polycystic ovary syndrome (PCOS) is a common endocrine condition affecting women of reproductive age. There are many typical signs and symptoms that allow for the diagnosis of PCOS depending on the criteria used. Interestingly, ethnicity influences the extent of these signs and symptoms; therefore, the frequency of symptoms varies between different countries and ethnic groups. The prevalence of this syndrome in Croatia is unknown, and it's clinical and biochemical characteristics have not yet been reported. During this study, we used the Rotterdam criteria to evaluate 365 Croatian women with PCOS, and compared them to 304 age matched controls to assess the clinical and biochemical abnormalities that occur in PCOS patients. The mean age of PCOS patients at presentation was 26.1 +/- 5.9 years and of controls were 28.0 +/- 4.2 years. Women with PCOS has significantly higher body mass index (BMI) than the control group, although in both groups most patients had normal weight (76.2% vs. 87.8%). Abdominal distribution of fat tissue was similar in both groups. Menstrual cycle abnormalities were observed in 90.7% of PCOS patients, and ultrasonographic appearance of polycystic ovaries was reported in 97.3% of PCOS cases. Nearly 75% of patients with PCOS had hirsutism and 49.6% had acne. We recorded significantly higher serum levels of luteinizing hormone (LH), total testosterone (TT), free testosterone (fT) and insulin, while the serum levels of sex hormone binding globuline (SHBG) and follicular stimulating hormone (FSH) were significantly lower than in the control group. Serum glucose values were not significantly different between the groups. In conclusion, chronic anovulation, hirsutism and ultrasound appearance of polycystic ovaries are the dominant features of PCOS in Croatian population. The majority of patients with PCOS had normal body weight. The incidence of insulin resistance in this group of patients is less than the previously described frequency in other populations of patients with PCOS and normal weight

Altered leptin, adiponectin, resistin and ghrelin secretion may represent an intrinsic polycystic ovary syndrome abnormality

The aim of the study was to investigate whether altered adipose tissue secretion of various adipokines is secondary to obesity, hyperandrogenism, and hyperinsulinemia or intrinsic to polycystic ovary syndrome (PCOS). This cross-sectional study included 151 women diagnosed with PCOS by the Rotterdam criteria and 95 healthy women matched by age, body mass index (BMI), and waist-to-hip ratio (WHR). Clinical, biochemical, and hormonal characteristics were assessed. Serum concentrations of ghrelin and adiponectin were found to be significantly lower and concentrations of leptin and resistin significantly higher in women with PCOS than in healthy women matched by age, BMI, and WHR. A PCOS diagnosis made the largest contribution to predicting serum levels of leptin, adiponectin, resistin, and ghrelin in all stepwise multiple regression models, which included PCOS diagnosis, BMI, WHR, luteinizing hormone, total testosterone, free testosterone and homeostatic model assessment of insulin resistance as independent predictors. Leptin, adiponectin, ghrelin and resistin levels may serve as independent biomarkers for the diagnosis of PCOS

Kliničke i biokemijske karakteristike bolesnica sa sindromom policističnih jajnika u Hrvatskoj populaciji

Polycystic ovary syndrome (PCOS) is a common endocrine condition affecting women of reproductive age. There are many typical signs and symptoms that allow for the diagnosis of PCOS depending on the criteria used. Interestingly, ethnicity influences the extent of these signs and symptoms; therefore, the frequency of symptoms varies between different countries and ethnic groups. The prevalence of this syndrome in Croatia is unknown, and it's clinical and biochemical characteristics have not yet been reported. During this study, we used the Rotterdam criteria to evaluate 365 Croatian women with PCOS, and compared them to 304 age matched controls to assess the clinical and biochemical abnormalities that occur in PCOS patients. The mean age of PCOS patients at presentation was 26.1 +/- 5.9 years and of controls were 28.0 +/- 4.2 years. Women with PCOS has significantly higher body mass index (BMI) than the control group, although in both groups most patients had normal weight (76.2% vs. 87.8%). Abdominal distribution of fat tissue was similar in both groups. Menstrual cycle abnormalities were observed in 90.7% of PCOS patients, and ultrasonographic appearance of polycystic ovaries was reported in 97.3% of PCOS cases. Nearly 75% of patients with PCOS had hirsutism and 49.6% had acne. We recorded significantly higher serum levels of luteinizing hormone (LH), total testosterone (TT), free testosterone (fT) and insulin, while the serum levels of sex hormone binding globuline (SHBG) and follicular stimulating hormone (FSH) were significantly lower than in the control group. Serum glucose values were not significantly different between the groups. In conclusion, chronic anovulation, hirsutism and ultrasound appearance of polycystic ovaries are the dominant features of PCOS in Croatian population. The majority of patients with PCOS had normal body weight. The incidence of insulin resistance in this group of patients is less than the previously described frequency in other populations of patients with PCOS and normal weight.indrom policističnih jajnika (PCOS) jedan je od najčešćih endokrinoloških poremećaja žena reprodukcijske dobi. Prezentira se, ovisno o kriterijima koji se koriste za postavljanje dijagnoze, s mnoštvom tipičnih znakova i simptoma. Pojavnost različitih znakova i simptoma razlikuje se među pojedinim etničkim skupinama. Prevalencija ovog sindroma u Hrvatskoj populaciji nije poznata, a njegove kliničke i biokemijske karakteristike do sada nisu istražene. U studiju smo uključili 365 žena s PCOS i usporedili ih s 304 zdrave žene koje su služile kao kontrole. Dijagnozu smo postavili na temelju Rotterdamskog konsezusa. Prosječna dob žena s PCOS iznosila je 26,1±5,9 godinu, a u kontrolnoj skupini 28,0±4,2 godinu. Žene s PCOS imale su značajno viši indeks tjelesne mase (ITM) u odnosu na kontrolnu skupinu, iako je većina žena uključenih u studiju imala normalnu tjelesnu težinu (76,2% naspram 87,8%). Učestalost abdominalne debljine bila je jednaka u obje ispitivane skupine. Abnormalnosti menstruacijskog ciklusa nađene su u 90,7% bolesnica s PCOS, a tipičan ultrazvučni nalaz policističnih jajnika u 97,3% slučajeva s PCOS. Gotovo 75% žena s PCOS imalo je hirzutizam, a 49,6% njih akne. Kod žena s PCOS nađene su značajno više vrijednosti luteinizirajućeg hormona (LH), ukupnog testosterone, slobodnog testosterone i inzulina, te snižene vrijednosti hormona koji veže spolne hormone (SHBG) i folikulostimulirajućeg hormona (FSH) u odnosu na kontrolnu skupinu. Vrijednosti glukoze, određene na tašte, nisu se značajno razlikovale među ispitivanim skupinama. Zaključno, kronična anovulacija, hirzutizam i ultrazvučni nalaz policističnih jajnika najčešće su značajke PCOS u Hrvatskoj populaciji. Većina žena s PCOS normalne je tjelesne težine. Učestalost inzulinske rezistencije u žena s PCOS i normalnom tjelesnom težinom u Hrvatskoj populaciji značajno je manja u odnosu na druge etničke skupine

Impact of Preanalytical and Analytical Methods on Cell-Free DNA Diagnostics

While tissue biopsy has for the longest time been the gold-standard in biomedicine, precision/personalized medicine is making the shift toward liquid biopsies. Cell-free DNA (cfDNA) based genetic and epigenetic biomarkers reflect the molecular status of its tissue-of-origin allowing for early and non-invasive diagnostics of different pathologies. However, selection of preanalytical procedures (including cfDNA isolation) as well as analytical methods are known to impact the downstream results. Calls for greater standardization are made continuously, yet comprehensive assessments of the impact on diagnostic parameters are lacking. This study aims to evaluate the preanalytic and analytic factors that influence cfDNA diagnostic parameters in blood and semen. Text mining analysis has been performed to assess cfDNA research trends, and identify studies on isolation methods, preanalytical and analytical impact. Seminal and blood plasma were tested as liquid biopsy sources. Traditional methods of cfDNA isolation, commercial kits (CKs), and an in-house developed protocol were tested, as well as the impact of dithiothreitol (DTT) on cfDNA isolation performance. Fluorimetry, qPCR, digital droplet PCR (ddPCR), and bioanalyzer were compared as cfDNA quantification methods. Fragment analysis was performed by qPCR and bioanalyzer while the downstream application (cfDNA methylation) was analyzed by pyrosequencing. In contrast to blood, semen as a liquid biopsy source has only recently begun to be reported as a liquid biopsy source, with almost half of all publications on it being review articles. Experimental data revealed that cfDNA isolation protocols give a wide range of cfDNA yields, both from blood and seminal plasma. The addition of DTT to CKs has improved yields in seminal plasma and had a neutral/negative impact in blood plasma. Capillary electrophoresis and fluorometry reported much higher yields than PCR methods. While cfDNA yield and integrity were highly impacted, cfDNA methylation was not affected by isolation methodology or DTT. In conclusion, NucleoSnap was recognized as the kit with the best overall performance. DTT improved CK yields in seminal plasma. The in-house developed protocol has shown near-kit isolation performance. ddPCR LINE-1 assay for absolute detection of minute amounts of cfDNA was established and allowed for quantification of samples inhibited in qPCR. cfDNA methylation was recognized as a stable biomarker unimpacted by cfDNA isolation method. Finally, semen was found to be an abundant source of cfDNA offering potential research opportunities and benefits for cfDNA based biomarkers development related to male reproductive health