Piotr Laudanski

Plasma apelin levels and apelin/APJ mRNA expression in patients with gestational diabetes mellitus

AIMS AND METHODS Apelin is a novel adipokine identified as an endogenous ligand of the G protein-coupled receptor APJ. In this study we compared plasma apelin concentrations in 101 patients with gestational diabetes (GDM) and 101 women with normal glucose tolerance (NGT) between 24 and 32 weeks of gestation (Group 1), as well as in 20 women with GDM and 16 subjects with NGT at term (Group 2). Apelin and APJ mRNA expression in subcutaneous adipose tissue (SAT), visceral adipose tissue (VAT) and placental tissue were also measured in Group 2, using quantitative real-time PCR. RESULTS There were no significant differences in plasma apelin levels between the women with GDM and NGT (Group 1: 1555.6 [1281.2-1804.2]pg/ml vs 1656.5 [1430.2-1852.1]pg/ml, Group 2: 1607.9 [1453.4-1768.7]pg/ml vs 1493.8 [1316.8-1956.7]pg/ml) nor in apelin and APJ mRNA expression in SAT, VAT and placental tissue. Apelin mRNA expression was approximately 10 fold higher in placental than in adipose tissue (p<0.0001). Apelin and APJ mRNA expression correlated significantly in SAT (R=0.45, p=0.03), VAT (R=0.69, p=0.003) and placental tissue (R=0.37, p=0.03). CONCLUSIONS No associations between circulating apelin or apelin/APJ mRNA expression and GDM or the indices of insulin resistance were noted in our study.

Expression of selected tumor suppressor and oncogenes in endometrium of women with endometriosis.

BACKGROUND It is becoming increasingly evident that the eutopic endometrium of women with endometriosis shows certain genetic alterations which are not found in the endometrium of disease-free women. The aim of the study was to compare the expression level of mammalian target of rapamycin (mTOR) tumor suppressor and oncogene-related genes in the endometrium of women with and without endometriosis as well as in ovarian endometriosis. METHODS A total of 81 regularly menstruating patients were recruited in the study. We applied the micro fluidic gene array to examine the expression of 15 human tumor suppressor and oncogenes in eutopic endometrium of 40 women with endometriosis and 41 controls without endometriosis. In 14 patients with endometriosis, gene expression was also studied in matched ovarian lesions. We studied the following genes: NF1, RHEB, mTOR, PTEN, TSC1, TSC2, KRAS, S6K1, TP53, EIF4E, LKB1, PIK3CA, BECN1, 4EBP1 and AKT1. Immunohistochemical studies were subsequently performed for selected proteins. RESULTS Of the 15 studied genes, we found significantly higher levels of oncogene AKT1 (P = 0.006) and tumor suppressor gene 4EBP1 (P = 0.01) mRNAs in the eutopic endometrium of women with endometriosis compared with control patients. Immunohistochemistry showed that 4EBP1 and AKT1 proteins were expressed in eutopic endometrium. CONCLUSIONS Our results suggest that up-regulation of AKT1 and 4EBP1 in eutopic endometrium may be associated with the pathogenesis of endometriosis, but their precise role remains to be established.

MicroRNAs expression profiling of eutopic proliferative endometrium in women with ovarian endometriosis

BackgroundThe eutopic endometrium of women with endometriosis, compared with disease-free individuals, contains certain molecular alterations, including the differential expression of microRNA (miRNA). The aim of the study was to compare the expression of the most relevant miRNAs in the eutopic endometrium of women with and without ovarian endometriosis.MethodsA total of 46 regularly menstruating patients, 21 patients with ovarian endometriosis and 25 controls, underwent surgery in the proliferative phase of the cycle. The eutopic endometrium was collected through aspirating biopsy prior to laparoscopy. Only patients with advanced (stage III and IV) histopathologically confirmed ovarian endometriosis were included. TaqMan MicroRNA Array Cards were applied to examine the expression of 667 human miRNAs in 10 patients with endometriosis and 10 controls. Custom-made, low-density real-time PCR arrays were used to confirm the expression of 15 selected molecules in 21 endometriosis patients and 25 disease-free individuals.ResultsOf 667 miRNAs, 2 were highly likely to be upregulated and 13 were downregulated in the eutopic endometrium of patients with endometriosis compared with the controls. Validation using real-time PCR showed that hsa-miR-483-5p (p = 0.012) and hsa-miR-629* (p = 0.02) are significantly downregulated in patients with endometriosis.ConclusionsChanges in the expression of select miRNAs might lead to or be a consequence of an early defect in the physiological activity of the proliferative endometrium, ultimately resulting in the overgrowth of this tissue outside the uterus.

A case of complete hypogonadotropic hypogonadism with a mutation in the gonadotropin-releasing hormone receptor gene

OBJECTIVE To screen for mutations in the GnRH receptor gene in a case of complete hypogonadotropic hypogonadism (HH) with GnRH resistance. DESIGN Case report. SETTING A university hospital. PATIENT(S) A male patient with the complete form of HH without anosmia. INTERVENTION(S) Physical examination and laboratory and genetic studies. MAIN OUTCOME MEASURE(S) Gonadotropins at the basal state and after GnRH administration and GnRH receptor DNA sequencing. RESULT(S) A novel missense mutation, localized in the first amino acid of the extracellular loop found in the heterozygous state, and another mutation, Arg(139)His (R139H), located in the conserved aspartate-arginine-serine motif at the junction of the third transmembrane and second intracellular loop of the GnRH receptor, were identified in the homozygous state. Pedigree studies reveal that both parents were heterozygous for R139H, while the mother carried the missense mutation at codon 1(M1T). CONCLUSION(S) GnRH receptor mutations may account for a larger proportion of cases of HH than previously thought. The phenotypic spectrum of HH seems to vary, and this heterogeneity may be related, at least in part, to the degree of impaired biological activity of the mutated GnRH receptor caused by the allelic type of mutations.

Circulating pro- and anti-inflammatory cytokines in Polish women with gestational diabetes.

In this study we measured serum concentrations of proinflammatory interleukin-6, interleukin-8, and interleukin-18 as well as anti-inflammatory interleukin-10 in 30 pregnant women with normal glucose tolerance, in 32 women with abnormal results of a 50-g glucose challenge test, and in 57 patients with gestational diabetes mellitus. Patients with gestational diabetes had significantly higher IL-6 (median 1.0 [0.7-1.5] vs. 0.7 [0.4-0.8] pg/ml, p=0.001), IL-8 (2.1 [1.1-4.2] pg/ml vs. 0.7 [0.4-0.9] pg/ml, p<0.0001), and IL-18 (249.3 [188.5-318.7] pg/ml vs. 186.7 [139.9-243.9] pg/ml, p=0.005) as well as lower IL-10 levels than healthy pregnant women (0.6 [0.5-1.5] pg/ml vs. 2.9 [1.8-3.2] pg/ml, p<0.0001). After adjusting for glucose, insulin, and BMI values, the differences in IL-8 and IL-18 became insignificant, whereas the differences in IL-6 and IL-10 levels remained highly significant (p<0.0001). The subjects with abnormal glucose challenge test results had higher IL-6 levels (0.9 [0.7-1.3] pg/ml, p=0.005) and similar levels of other cytokines as compared with the women with normal glucose tolerance. Our results suggest an impaired balance between circulating pro- and anti-inflammatory cytokines in patients with gestational diabetes; however, a significant contribution of maternal obesity to the increased levels of IL-8 and IL-18 should be underlined.

Serum irisin concentration in women with gestational diabetes

Abstract Irisin is a novel myokine and adipokine which induces an increase in total body energy expenditure, improving insulin sensitivity and glucose tolerance in experimental animals. In the present study, serum irisin concentration was measured by an enzyme immunoassay in 130 women with gestational diabetes mellitus (GDM) and 140 BMI-matched patients with normal glucose tolerance (NGT). Median irisin level was significantly lower in the patients with GDM than in the NGT subjects (1703.3 [1354.8–2097.9 ng/ml] versus 1873.8 [1519.8–2294.8 ng/ml], p = 0.01); however, 3 months after childbirth its concentrations did not differ markedly between the two groups (1165.9 [872.1–1497.5] ng/ml versus 1139.0 [984.0–1376.7] ng/ml). In the whole group, irisin concentration correlated negatively with 2 h glucose level (R = −0.14, p = 0.03). In the women with NGT, irisin concentration correlated positively with ISOGTT (R = 0.22, p = 0.04) and the disposition index (DI120) (R = 0.24, p = 0.03), as well as negatively with 2 h insulin level (R = −0.23, p = 0.03) and HOMA-IR (R = −0.24, p = 0.02). Multiple regression analysis revealed that 2 h glucose and DI120 were the only variables significantly influencing serum irisin (β = 0.158, p = 0.03 and β = 0.159, p = 0.02, respectively). Our results suggest that serum irisin concentration increases markedly in pregnant women, but this increase seems to be significantly lower in patients with GDM. Chinese abstract 鸢尾素是一种新发现的肌细胞因子和脂肪因子，在动物实验中，可以提高全身能量消耗，提高胰岛素敏感性和糖耐量。本研究通过酶免测定法测定了130名妊娠糖尿病(GDM)妇女和140名BMI值相匹配的糖耐量正常(NGT)妇女血清鸢尾素浓度。GDM患者中位血清鸢尾素浓度较NGT妇女明显降低(1703.3 [1354.8–2097.9 ng/ml] 比1873.8 [1519.8–2294.8 ng/ml], p=0.01)；而在分娩三个月后两组妇女血清鸢尾素浓度并无明显差异(1165.9 [872.1–1497.5] ng/ml 比 1139.0 [984.0–1376.7]ng/ml)。所有受试对象的血清鸢尾素浓度均与餐后2h 血糖呈负相关(R=－0.14, p=0.03)。NGT妇女血清鸢尾素浓度与处置指数（DI120）(R=0.24, p=0.03)和ISOGTT(R=0.22, p=0.04)正相关，与2h胰岛素水平(R=－0.23, p=0.03)和HOMA-IR (R=﹣0.24, p=0.02)负相关。多元回归分析显示只有餐后2h血糖与DI120是显著影响血清鸢尾素浓度的变量(分别为β=0.158, p=0.03 和β=0.159, p=0.02)。研究结果显示妊娠妇女血清鸢尾素浓度明显增高，而在GDM妇女这种升高值则明显降低。

Chemokine growth-regulated-α: a possible role in the pathogenesis of endometriosis

Endometriosis is a common disease in women at reproductive age. We investigated the concentration of neutrophil-activating factor (growth-regulated gene-α; GRO-α) (a member of the chemokine family), in peritoneal fluid of infertile women with or without endometriosis. Peritoneal fluid was obtained laparoscopically from 22 women with and 21 without visible endometriotic lesions. GRO-α concentration was measured by the use of an ELISA kit. Median concentration of GRO-α was 87.65 ± 56.19 pg/ml in the study group and 60.72 ± 11.98 pg/ml in the control group. The distribution of data differed from normal, therefore logarithmic transformation of data was performed. Concentration of GRO-α was significantly higher in the peritoneal fluid of women with endometriosis when compared with controls (p = 0.05). No correlation between concentration of GRO-α, stage of endometriosis, duration of infertility or sex steroid hormone levels was found. The study shows that GRO-α may play a role in the pathogenesis of endometriosis, possibly by chemoattraction and activation of neutrophils present in higher numbers in the peritoneal fluid of women with endometriosis. It is also feasible that the angiogenic properties of GRO-α might prompt the progression of endometriotic lesions.

Matrix metalloproteinase-13 and membrane type-1 matrix metalloproteinase in peritoneal fluid of women with endometriosis

Objective. It is becoming more and more evident that different types of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in the pathogenesis of endometriosis. The aim of the present study was to measure levels of the active forms of MMP-13 and membrane type-1 matrix metalloproteinase (MT1-MMP)/MMP-14 as well as TIMP-2 in the peritoneal fluid of women with endometriosis. Study design. We determined the levels of the active forms MMP-13 and MT1-MMP/MMP-14 as well as TIMP-2 in the peritoneal fluid of 20 women with endometriosis and 18 controls by different types of enzyme-linked immunosorbent assay. Results. We found that the concentrations (mean ± standard deviation) of total active MMP-13 and endogenous active MT1-MMP/MMP-14 in the peritoneal fluid of patients with endometriosis were 1.69 ± 0.67 and 3.12 ± 1.07 ng/ml, respectively. In control women the corresponding values were 3.02 ± 0.43 and 4.45 ± 1.03 ng/ml. The differences were statistically significant (p < 0.0001 and p < 0.0004 for MMP-13 and MMP-14, respectively). Levels of TIMP-2 did not differ significantly. Conclusions. Decreased concentrations of active MMP-13 and MT1-MMP/MMP-14 may imply that the proteolytic activity of the peritoneal milieu of women with endometriosis is disturbed, which may have implications in the pathogenesis of the disease.

Increased Maternal and Cord Blood Betatrophin in Gestational Diabetes.

Aim The aim of the study was to compare maternal and cord blood levels of betatrophin – a new peptide potentially controlling beta cell growth - as well as in its mRNA expression in subcutaneous adipose tissue, visceral adipose tissue and placental tissue obtained from pregnant women with normal glucose tolerance (NGT) and gestational diabetes (GDM). Methods Serum betatrophin and irisin concentrations were measured by ELISA in 93 patients with GDM and 97 women with NGT between 24 and 28 week of gestation. Additionally, maternal and cord blood betatrophin and irisin, as well as their genes (C19orf80 and Fndc5) expression were evaluated in 20 patients with GDM and 20 women with NGT at term. Results In both groups, serum betatrophin concentrations were significantly higher in the patients with GDM than in the controls (1.91 [1.40-2.60] ng/ml vs 1.63 [1.21-2.22] ng/ml, p=0.03 and 3.45 [2.77-6.53] ng/ml vs 2.78 [2.16-3.65] ng/ml, p=0.03, respectively). Cord blood betatrophin levels were also higher in the GDM than in the NGT group (20.43 [12.97-28.80] ng/ml vs 15.06 [10.11-21.36] ng/ml, p=0.03). In both groups betatrophin concentrations in arterial cord blood were significantly higher than in maternal serum (p=0.0001). Serum irisin levels were significantly lower in the patients with GDM (1679 [1308-2171] ng/ml) than in the healthy women between 24 and 28 week of pregnancy (1880 [1519-2312] ng/ml, p=0.03). Both C19orf80 and Fndc5 mRNA expression in fat and placental tissue did not differ significantly between the groups studied. Conclusions Our results suggest that an increase in maternal and cord blood betatrophin might be a compensatory mechanism for enhanced insulin demand in GDM.

Development of surface plasmon resonance imaging biosensors for detection of ubiquitin carboxyl-terminal hydrolase L1.

We have developed a new method for highly selective determination of the ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) concentration using a surface plasmon resonance imaging (SPRI) technique and two different biosensors. UCH-L1 was captured from a solution by immobilized specific rabbit monoclonal antibody or specific LDN-57444 inhibitor due to formation of receptor-UCH-L1 complex on the biosensor surface. The analytically useful dynamic response range of both biosensors is between 0.1 and 2.5ng/ml. The detection limit is 0.06ng/ml for the biosensor with antibody and 0.08ng/ml for the biosensor with inhibitor. Biosensors based on both antibody and inhibitor were found to be suitable for quantitative determination of the UCH-L1 and exhibit good tolerance to the potential interferents. Both biosensors gave comparable results in the range of 0 to 0.20ng/ml for plasma samples and 0.30 to 0.49ng/ml for cerebrospinal fluid samples. To validate the new methods, comparative determination of UCH-L1 by the commercial enzyme-linked immunosorbent assay (ELISA) kit was performed. In general, in terms of UCH-L1 concentration, a good correlation between SPRI and ELISA was found. The developed biosensors can be used successfully for the determination of UCH-L1 in body fluids.