Wnt4, Wnt6 and ß-catenin expression in human placental tissue - is there a link with first trimester miscarriage? Results from a pilot study.

BACKGROUND: Demystifying the events around early pregnancy is challenging. A wide network of mediators and signaling cascades orchestrate the processes of implantation and trophoblast proliferation. Dysregulation of these pathways could be implicated in early pregnancy loss. There is accumulating evidence around the role of Wnt pathway in implantation and early pregnancy. The purpose of this study was to explore alterations in the expression of Wnt4, Wnt6 and ß-catenin in placental tissue obtained from human first trimester euploid miscarriages versus normally developing early pregnancies. METHODS: The study group consisted of first trimester miscarriages (early embryonic demises and incomplete miscarriages) and the control group of social terminations of pregnancy (TOPs). The placental mRNA expression of Wnt4, Wnt6 and ß-catenin was studied using reverse transcription PCR and real time PCR. Only euploid conceptions were included in the analysis. RESULTS: Wnt4 expression was significantly increased in placental tissue from first trimester miscarriages versus controls (p = 0.003). No significant difference was documented in the expression of Wnt6 (p = 0.286) and ß-catenin (p = 0.793). There was a 5.1fold increase in Wnt4 expression for early embryonic demises versus TOPs and a 7.6fold increase for incomplete miscarriages versus TOPs - no significant difference between the two subgroups of miscarriage (p = 0.533). CONCLUSIONS: This is, to our knowledge, the first study demonstrating significant alteration of Wnt4 expression in human placental tissue, from failed early pregnancies compared to normal controls. Undoubtedly, a more profound study is needed to confirm these preliminary findings and explore Wnt mediators as potential targets for strategies to predict and prevent miscarriage.

Association of Calpain (CAPN) 10 (UCSNP-43, rs3792267) gene polymorphism with elevated serum androgens in young women with the most severe phenotype of polycystic ovary syndrome (PCOS).

OBJECTIVES: To highlight a possible association of Calpain (CAPN 10) gene UCSNP-43 polymorphism with hormonal and metabolic traits of young women with different phenotypes of polycystic ovary syndrome (PCOS). DESIGN: PCOS women were genotyped for the CAPN 10 gene UCSNP-43 polymorphism. A comparison of clinical and biochemical features of women with PCOS stratified on the basis of the CAPN 10 gene UCSNP-43 variants was assessed. METHODS: Anthropometric, hormonal and biochemical measurements were carried out in 668 PCOS women and 200 healthy controls. Subjects were also genotyped for the CAPN 10 gene UCSNP-43 polymorphism. The genotype frequency distributions between groups and controls were compared using the chi-square test. The association of the polymorphism with the clinical and biochemical features of the study cohort was estimated as well. RESULTS: No association of the frequency of CAPN 10 gene UCSNP-43 polymorphism with PCOS was detected. No association of the polymorphism with the anthropometric, biochemical and hormonal features was detected both in PCOS and control women. The polymorphism was associated with serum Δ4 androstenedione (p = 0.018), as well as with 17-OH progesterone (17-hydroxyprogesterone) among women with PCOS phenotype A (p = 0.012). CONCLUSIONS: CAPN 10 gene polymorphism UCSNP-43 is deprived of a metabolic contribution to cardiovascular disease (CVD). However, due to its association with androgen excess in phenotype A, CAPN 10 gene polymorphism UCSNP-43 could be used as a genetic marker for CVD in young PCOS women.

Placental mRNA Expression of Neurokinin B Is Increased in PCOS Pregnancies with Female Offspring.

Current research suggests that polycystic ovary syndrome (PCOS) might originate in utero and implicates the placenta in its pathogenesis. Kisspeptin (KISS1) and neurokinin B (NKB) are produced by the placenta in high amounts, and they have been implicated in several pregnancy complications associated with placental dysfunction. However, their placental expression has not been studied in PCOS. We isolated mRNA after delivery from the placentae of 31 PCOS and 37 control women with term, uncomplicated, singleton pregnancies. The expression of KISS1, NKB, and neurokinin receptors 1, 2, and 3 was analyzed with real-time polymerase chain reaction, using ß-actin as the reference gene. Maternal serum and umbilical cord levels of total testosterone, sex hormone-binding globulin (SHBG), free androgen index (FAI), androstenedione, dehydroepiandrosterone sulfate (DHEAS), Anti-Mullerian hormone (AMH), and estradiol were also assessed. NKB placental mRNA expression was higher in PCOS women versus controls in pregnancies with female offspring. NKB expression depended on fetal gender, being higher in pregnancies with male fetuses, regardless of PCOS. NKB was positively correlated with umbilical cord FAI and AMH, and KISS1 was positively correlated with cord testosterone and FAI; there was also a strong positive correlation between NKB and KISS1 expression. Women with PCOS had higher serum AMH and FAI and lower SHBG than controls. Our findings indicate that NKB might be involved in the PCOS-related placental dysfunction and warrant further investigation. Studies assessing the placental expression of NKB should take fetal gender into consideration.

Expression stability of ACTB, 18S, and GAPDH in human placental tissues from subjects with PCOS and controls: GAPDH expression is increased in PCOS.

PURPOSE: The aim was to assess the expression stability of three commonly used reference genes, namely, ß-actin (ACTB), 18S ribosomal RNA (18S), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in placental tissue obtained from pregnant women with polycystic ovary syndrome (PCOS) and healthy controls. METHODS: mRNA was isolated after delivery from the placentae of 10 PCOS and 10 control women with term, uncomplicated, singleton pregnancies. The expression of ACTB, 18S, and GAPDH was analyzed using real-time polymerase chain reaction (RT-PCR). Gene expression stability was evaluated with the RefFinder, GeNorm, Normfinder, BestKeeper, and Delta-Ct tools. RESULTS: ACTB was ranked as the most stably expressed gene, followed by 18S. The expression of GAPDH varied considerably in both studied groups, while it was increased in PCOS versus controls (5.3-fold, p < 0.05). CONCLUSIONS: ACTB is an appropriate reference gene for placental gene expression studies in women with PCOS, whereas GAPDH is unfit for such a role, as its placental expression is increased in PCOS.

Adequate vitamin D supplementation does not ameliorate bone loss following long limb-biliopancreatic diversion in morbidly obese women.

OBJECTIVES: The objective of this study is to investigate the effect of adequate vitamin D supplementation on bone mineral density (BMD) following long limb-biliopancreatic diversion (LL-BPD), a malabsorptive bariatric operation. BACKGROUND: Marked weight loss following bariatric surgery is associated with significant decrease in BMD, attributed to the weight loss and to nutritional, mineral, and vitamin D deficiencies resulting in secondary hyperparathyroidism. METHODS: Two groups, of 35 and 37 healthy, obese (BMI, 50.4 + 6.6 and 46.5 + 4.8 g/cm2), premenopausal, normally menstruating women underwent LL-BPD. Both groups received high-calcium diets, 600 IU of vitamin D, and 1000 mg elemental calcium daily, while group B received an extra dose of vitamin D (10,000 IU/day) during the first postoperative month, followed by dose adjustment in order to maintain 25OHD concentration higher than 30 µg/L. Areal BMD (aBMD) was measured at the lumbar spine preoperatively and 1 year postoperatively. RESULTS: One year postoperatively, BMI decreased by approximately 19 kg/m2 in both groups, while 25-OH-vitamin D levels did not change in group A (18.7 + 9.1 to 20.2 + 13.0 µg/L, (p = 0.57)) and increased in group B (15.58 ± 5.73 to 52.97 ± 15.46 µg/L, (p = < 0.001). PTH levels increased in group A (from 38.5 ± 12.2 to 51.2 ± 32.8 pg/ml) (p = 0.047) and decreased in group B (from 51.61 ± 18.7 to 45.1 ± 17.8 pg/ml) (p = 0.042). Lumbar spine aBMD decreased similarly in both groups (p = 0.311, for the comparison between groups) from 1.198 + 0.14 to 1.103 + 0.15 g/cm2 in group A (p < 0.001) and from 1.157 + 0.14 to 1.076 + 0.14 g/cm2 in group B (p < 0.001) and Z-score from 0.93 + 0.97 to 0.19 + 1.02, (p < 0.001) and from 1.15 + 1.29 to 0.419 + 1.28, (p < 0.001), respectively. CONCLUSIONS: LL-BPD leads to similar and significant bone mass reduction 1 year postoperatively, irrespective of adequate vitamin D replacement and in the absence of secondary hyperparathyroidism.

Current Practice in FFP Preparation and Use in Greece: A National Survey

Objective: Fresh frozen plasma (FFP) transfusion is widely used in modern clinical settings. Practices regarding its use vary due to lack of guidelines from randomized trials. The aim of this study was to assess both the current practices regarding FFP production, use, and wastage and the implementation of quality control (QC), female donor plasma production policies, and use of pharmaceutical hemostatic agents in Greece. Materials and Methods: The study was conducted during February-April 2018. For the first part of the study, data including FFP transfusion indication, hospital department, diagnosis, FFP units/transfusion episode, ABO compatibility, blood donor's sex, and reasons for discarding were collected. For the second part, questionnaire data were analyzed. Results: According to data from 20 Greek hospitals, 12655 FFP units were transfused to 2700 patients during 5069 transfusion episodes in the studied period of time. Most patients were hospitalized in internal medicine, general surgery, and intensive care unit departments. Each patient received on average 4.69 units (2.5 units/episode). Transfusion requests were in accordance with international guidelines in 63.44% of cases and 99.04% of the units were given to ABO-identical patients. Main reasons for discarding included failure to meet quality requirements (30.06%), female donors (22.17%), and other causes (27.26%). Among 96.9% of all transfusion services across the country, 28.26% perform QC according to the directions of the European Directorate for the Quality of Medicines & Health Care and 68.83% discard plasma from female donors. Pharmaceutic hemostatic agents are used in 37.23% of the hospitals. Conclusion: This is the first national survey regarding FFP production and transfusion in Greece. Staff of internal medicine, general surgery, and ICU departments, where most FFP-transfused patients are hospitalized, should be regularly involved in training on contemporary transfusion guidelines. Upcoming centralization of FFP production and inventory management could help in homogenizing practices regarding FFP use and improve product quality. Strengthening the use of pharmaceutic hemostatic agents could improve patients' management.

No association of the G972S polymorphism of the insulin receptor substrate-1 gene with polycystic ovary syndrome in lean PCOS women with biochemical hyperandrogenemia.

PURPOSE: The aim of the present study was to determine the prevalence and association of the G972S polymorphism of the insulin receptor substrate-1 gene (IRS-1 G972S SNP) with polycystic ovary syndrome (PCOS) and insulin resistance-related traits in a distinct phenotypic group of lean PCOS women with biochemical hyperandrogenemia, excluding obesity, which is considered to be an aggravating parameter of insulin resistance. METHODS: The study included 162 women with PCOS and 122 regularly menstruating, ovulatory women as controls. Physical measurements included weight, height, fat-free mass, fat mass, systolic and diastolic blood pressure and resting heart rate. Biochemical parameters included the serum testosterone, free testosterone, androstenedione, total cholesterol, triglycerides, HDL and LDL cholesterol and glucose levels. Insulin resistance was assessed by determining fasting insulin levels, fasting glucose levels, the fasting glucose/insulin ratio, as well as the HOMA and QUICKI indexes. All DNA samples were genotyped by a PCR-restriction fragment length polymorphism (RLFP) assay. RESULTS: No association of the genotype frequencies of the G972S polymorphism in insulin receptor substrate-1 gene (IRS-1 G972S SNP) with PCOS phenotype and insulin resistance was detected. CONCLUSION: The G972S polymorphism of the IRS-1 gene should not be viewed as major contributor to the development of PCOS or as a causative variant for insulin resistance.

Reduced Foxo3a, FoxL2, and p27 mRNA expression in human ovarian tissue in premature ovarian insufficiency.

PURPOSE: Previous studies have suggested that deletion of Foxo3a, FoxL2, PTEN, p27, and AMH leads to early exhaustion of the primordial follicle pool and premature ovarian insufficiency (POI) in transgenic mice. Our aim was to assess for the first time, to our knowledge, messenger RNA (mRNA) expression of these genes and AMHR2 in human ovarian tissue from women with POI. We hypothesized that these genes would be underexpressed in POI women compared with healthy controls. METHODS: mRNA levels were evaluated by quantitative reverse transcription-polymerase chain reaction and real-time polymerase chain reaction in cortical ovarian tissue obtained by laparoscopy from Caucasian Greek women with POI (n = 5) and healthy women with normal menstruation (n = 6). Morphological analysis of the ovarian biopsies was also performed to assess the presence of primordial or other types of growing follicles. RESULTS: Ovarian tissue from POI patients showed lower Foxo3a, FoxL2, and p27 mRNA expression compared with controls (p = 0.017, p = 0.017, and p = 0.030, respectively). mRNA expression of AMH, PTEN, and AMHR2 was reduced in ovarian biopsies from POI patients as well. However, these differences were not statistically significant (p = 0.143, p = 0.247, and p = 0.662, respectively). Morphological analysis showed complete lack of follicular structures in all POI biopsies. CONCLUSIONS: Our findings suggest a possible role of Foxo3a, FoxL2, and p27 in the pathogenesis of human POI, which may prove to be of great diagnostic-therapeutic value. Further larger studies are needed to identify a similar pattern for AMH, PTEN, and AMHR2 and to investigate gene expression at a protein level.

Correction to: Reduced Foxo3a, FoxL2, and p27 mRNA expression in human ovarian tissue in premature ovarian insufficiency.

The original version of this article, published 21 October 2019, unfortunately contained a mistake. The presentation of Fig. 1 was incorrect. The corrected figure is given below.

Delayed diagnosis of disorder of sex development (DSD) due to P450 oxidoreductase (POR) deficiency.

CASE PRESENTATION: A 36-year old man, operated on for cryptorchidism at the age of 8 years, was referred to the Outpatient Clinic of Reproductive Endocrinology for investigation of infertility. Clinical examination revealed ambiguous genitalia: penis 4-5 cm, testicular volume 2-3 ml, hypospadias, hypertrophic foreskin and scrotum bifida. Mild hypertension was confirmed. No skeletal malformations were detected. DESIGN: Hormonal and electrolytic determinations as well as semen analysis were conducted. PCR of the coding regions of 17-hydroxylase/17,20 lyase (P450c17) and of P450 oxidoreductase (POR) genes was also performed. RESULTS: Normal levels of electrolytes, low levels of androgens, high levels of gonadotropins and 17-hydroxyprogesterone as well as azoospermia were detected. Karyotype was shown to be 46,XY. Both hCG and ACTH stimulation significantly increased 17-hydroxyprogesterone with no increase in androgens. The diagnosis was congenital adrenal hyperplasia with apparent combined P450c17 and P450c21 deficiency due to mutations in the POR gene. Sequencing of the POR gene revealed: one deletion in exon 12 (Del 1696_1698delGTC >del531Valine) and one missense mutation in exon 7 (A259G) as well as two polymorphisms: rs1057868 (C/T A503V) and rs1057870 (G/A S572S) in exons 12 and 13, respectively. No nucleotide changes were detected in the 8 exons of P450c17. CONCLUSIONS: Molecular findings were consistent with the diagnosis of P450 oxidoreductase deficiency. Despite this severe deficiency, skeletal malformations simulating Antley-Bixler syndrome, which usually characterize the most severe forms, were not confirmed. This discrepancy could be attributed to the differential impact of a POR variant on each one of the P450 enzymes.

Increased frequency of the anti-mullerian-inhibiting hormone receptor 2 (AMHR2) 482 A>G polymorphism in women with polycystic ovary syndrome: relationship to luteinizing hormone levels.

CONTEXT: The polycystic ovary syndrome (PCOS) is a common and complex disease without a clear pattern of inheritance. Anti-Müllerian hormone (AMH) has an inhibitory effect on FSH-stimulated follicle growth. Serum AMH levels are higher in women with PCOS than in normo-ovulatory women. The elevated AMH levels may reflect abnormalities in AMH signaling. OBJECTIVE: The purpose of this study was to evaluate the association of the anti-Müllerian hormone receptor 2 (AMHR2) -482 A>G polymorphism (rs2002555) with the pathophysiology of PCOS. DESIGN: AMHR2 -482 A>G polymorphism genotyping were performed in a large cohort of women with PCOS and in a healthy control group. SETTING/SUBJECTS: A total of 858 Caucasian Greek women with PCOS and 309 healthy control women were studied. INTERVENTIONS: Genotyping and hormonal measurements were preformed. MAIN OUTCOME MEASURES: Hormone levels in women with PCOS were analyzed. RESULTS: The AMHR2 polymorphism was more common in women with PCOS than in control women (P = .026). Homozygous AMHR2 -482 A>G gene polymorphisms (GG) were associated with decreased levels of LH (P = .003) and lower LH to FSH ratios (P = .01) in women with PCOS, as well as with lower prolactin levels (P = .004). No other associations related to AMHR2 -482 A>G polymorphisms were observed in women with PCOS or control women. CONCLUSION: In this study, the role of the AMHR2 -482 A>G gene polymorphism in the pathogenesis of PCOS was suggested by the association of the variant with PCOS risk. Thus, further research is needed to elucidate a possible association of the AMHR2 -482 A>G gene polymorphism with AMH signaling and impaired ovarian function and its clinical significance in women with PCOS.

Comparative functional analysis of two fibroblast growth factor receptor 1 (FGFR1) mutations affecting the same residue (R254W and R254Q) in isolated hypogonadotropic hypogonadism (IHH).

FGFR1 mutations have been identified in both Kallmann syndrome and normosmic HH (nIHH). To date, few mutations in the FGFR1 gene have been structurally or functionally characterized in vitro to identify molecular mechanisms that contribute to the disease pathogenesis. We attempted to define the in vitro functionality of two FGFR1 mutants (R254W and R254Q), resulting from two different amino acid substitutions of the same residue, and to correlate the in vitro findings to the patient phenotypes. Two unrelated GnRH deficient probands were found to harbor mutations in FGFR1 (R254W and R254Q). Mutant signaling activity and expression levels were evaluated in vitro and compared to a wild type (WT) receptor. Signaling activity was determined by a FGF2/FGFR1 dependent transcription reporter assay. Receptor total expression levels were assessed by Western blot and cell surface expression was measured by a radiolabeled antibody binding assay. The R254W maximal receptor signaling capacity was reduced by 45% (p<0.01) while R254Q activity was not different from WT. However, both mutants displayed diminished total protein expression levels (40 and 30% reduction relative to WT, respectively), while protein maturation was unaffected. Accordingly, cell surface expression levels of the mutant receptors were also significantly reduced (35% p<0.01 and 15% p<0.05, respectively). The p.R254W and p.R254Q are both loss-of-function mutations as demonstrated by their reduced overall and cell surface expression levels suggesting a deleterious effect on receptor folding and stability. It appears that a tryptophan substitution at R254 is more disruptive to receptor structure than the more conserved glutamine substitution. No clear correlation between the severity of in vitro loss-of-function and phenotypic presentation could be assigned.

Nrf2 is commonly activated in papillary thyroid carcinoma, and it controls antioxidant transcriptional responses and viability of cancer cells.

CONTEXT: The antioxidant transcription factor NFE2-related factor 2 (Nrf2), encoded by NFE2L2, has been implicated as mediator of thyroid cancer cell line resistance to proteasome inhibitors. However, the activity status of the Nrf2 pathway in human thyroid cancer remains unknown. OBJECTIVE: The aims of this study were assessment of the activity status of the Nrf2 pathway in papillary thyroid carcinoma (PTC) and investigation of its role(s) in antioxidant transcriptional responses and viability of cancer cells. DESIGN AND SETTING: We conducted retrospective immunohistochemical analyses of PTC specimens, adjacent normal tissue, and benign lesions; assays of viability and gene expression in the PTC cell lines K1 and TPC-1 after genetic/pharmacological manipulation of Nrf2; and DNA sequencing at an academic medical center. PATIENTS: The study included 42 PTC and 42 benign lesions (24 adenomas and 18 nodular hyperplasias). MAIN OUTCOME MEASURES: We assessed the abundance of Nrf2, Nqo1, Keap1, and 4HNE; cell line viability and mRNA expression of Nrf2, Nqo1, and Trdx1; and the sequence of NFE2L2, KEAP1, and BRAF. RESULTS: Nrf2 and its target Nqo1 were undetectable in normal tissue; their levels were significantly higher in PTC than in benign lesions (P < .0001 and P = .024, respectively). The Nrf2 inhibitor Keap1 was variably abundant in PTC, and its levels did not correlate with Nrf2 (P = .37), arguing against decreased levels as the mechanism for Nrf2 activation. The oxidized lipid 4HNE was more abundant in PTC than normal tissue (P < .001), indicating oxidative stress. Nrf2 mediated transcriptional antioxidant responses in both the PTC cell lines K1 and TPC-1 and in the nontransformed cell line TAD2, but it conferred a viability advantage specifically in the PTC cell lines. CONCLUSIONS: The high activity of Nrf2 in PTC warrants further exploration of this pathway's potential diagnostic, prognostic, and/or therapeutic utility in PTC.

The expression of omental 11ß-HSD1 is not increased in severely obese women with metabolic syndrome.

OBJECTIVE: Plasma cortisol in obese subjects does not differ from that in normoweight subjects. Extra-adrenal cortisol production by 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) can result in local hypercortisolemia. The aim of the present study was to examine the role of visceral hypercortisolemia in the development of metabolic syndrome in severe obesity. METHODS: Eight lean women during hysterectomy (controls) and 19 severely obese women during bariatric surgery were studied, 8 without metabolic syndrome (OM- group) and 11 with it (OM+ group). Biopsies of omental and subcutaneous fat were performed in the severely obese women during surgery, but only omental biopsies in the controls. Expression of 11ß-HSD1, glucocorticoid receptor α (GRα) and glucocorticoid receptor ß (GRß) was evaluated using real-time PCR. RESULTS: Omental 11ß-HSD1 expression was different between groups (one-way ANOVA, p < 0.01). Post-hoc analysis revealed that mean omental 11ß-HSD1 mRNA levels were higher in the OM- group compared to controls, whereas they were similar when comparing the OM+ group with lean controls. Expression of 11ß-HSD1 in subcutaneous fat was not different between OM+ and OM- groups. GRα expression in omental fat did not differ among groups or between omental and subcutaneous fat in severely obese patients. An expression of GRß was not detected. CONCLUSION: Contrary to our original hypothesis, omental 11ß-HSD1 expression is not increased in the OM+ group.

Increased frequency of the DI genotype of the angiotensin-I converting enzyme and association of the II genotype with insulin resistance in polycystic ovary syndrome.

OBJECTIVE: The polycystic ovary syndrome (PCOS) is a common and complex disease with unclear pattern of inheritance, characterized by an androgen excess, while hyperinsulinemia and insulin resistance (IR) are common features of the syndrome. The angiotensin I converting enzyme (ACE) insertion (I)/deletion (D) gene polymorphism was proved to be involved in many pathophysiological conditions, including hypertension and IR. DESIGN: The purpose of this study was to evaluate the involvement of the ACE gene polymorphism in the pathogenesis of PCOS. METHODS: In a case-control association study involving 801 PCOS women and 266 healthy controls, hormonal determinations and ACE polymorphism genotyping were performed. The PCOS women were classified into three groups: Group A presented biochemical hyperandrogenism, combined with anovulation and polycystic ovarian morphology; Group B, clinical hyperandrogenism combined with anovulation and polycystic ovarian morphology; and Group C, chronic anovulation and polycystic ovarian morphology. RESULTS: A significant increase in the frequency of the DI genotype of the ACE polymorphism was detected in PCOS women as a whole (P=0.035), in PCOS Group A (P=0.039) and Group B (P=0.010), while there was no difference in Group C (P=0.939). Significant difference was also observed in hyperandrogenic PCOS women as a whole (Group A+B) (P=0.017). The II genotype was positively correlated with HOMA-IR and QUICKI and with fasting insulin and glucose/insulin ratio in these groups. CONCLUSIONS: The association study of the ACE I/D polymorphism in PCOS women demonstrates an increase in the DI genotype incidence and an association of the II genotype with IR.

PKG-I inhibition attenuates vascular endothelial growth factor-stimulated angiogenesis.

Vascular endothelial growth factor (VEGF) stimulates nitric oxide (NO) production, which mediates many of its angiogenic actions. However, the angiogenic pathways that operate downstream of NO following VEGF treatment are not well characterized. Herein, we used DT-2 and DT-3, two highly selective cGMP-dependent protein kinase I peptide inhibitors to determine the contribution of PKG-I in VEGF-stimulated angiogenesis. Incubation of chicken chorioallantoic membranes (CAM) with PKG-I peptide inhibitors decreased vascular length in a dose-dependent manner, with DT-3 being more effective than DT-2. Moreover, inhibition of PKG-I with DT-3 abolished the angiogenic response elicited by VEGF in the rabbit eye cornea. PKG-I inhibition also blocked VEGF-stimulated vascular leakage. In vitro, treatment of cells with VEGF stimulated phosphorylation of the PKG substrate VASP through VEGFR2 activation; the VEGF-stimulated VASP phosphorylation was reduced by DT-2. Pre-treatment of cells with DT-2 or DT-3 inhibited VEGF-stimulated mitogen-activated protein kinase cascades (ERK1/2 and p38), growth, migration and sprouting of endothelial cells. The above observations taken together identify PKG-I as a downstream effector of VEGFR2 in EC and provide a rational basis for the use of PKG-I inhibitors in disease states characterized by excessive neovascularization.

Association of the 17-hydroxysteroid dehydrogenase type 5 gene polymorphism (-71A/G HSD17B5 SNP) with hyperandrogenemia in polycystic ovary syndrome (PCOS).

OBJECTIVE: To evaluate the association of an activating single-nucleotide polymorphism (SNP) at position -71 of the promoter of 17beta-hydroxysteroid dehydrogenase type 5 gene (-71A/G HSD17B5 SNP) and polycystic ovary syndrome (PCOS) in a well characterized cohort of caucasian PCOS women with biochemical hyperandrogenemia. DESIGN: The PCOS patients and unrelated healthy control subjects were genotyped for the -71A/G HSD17B5 SNP. The acquired genotypic data was tested for association with PCOS and other quantitative phenotypic traits of the syndrome in PCOS patients. SETTING: Subjects were recruited from the Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, at the University Hospital of Patras, Greece. Genotyping and biochemical determinations took place at the Laboratory of Molecular Endrocinology, University of Patras Medical School, Rion, Greece. PATIENT(S): Participants comprised 150 caucasian Greek PCOS women with biochemical hyperandrogenism and chronic anovulation and polycystic ovarian morphology on ultrasound and 51 healthy control subjects. MAIN OUTCOME MEASURE(S): HSD17B5 genotype, serum testosterone, serum androstenedione. RESULT(S): No association of the -71A/G HSD17B5 SNP with PCOS was detected. However, the -71G HSD17B5 variant was associated with increased serum testosterone levels and decreased androstenedione/testosterone ratio. CONCLUSION(S): The -71G HSD17B5 variant is not a major component of the molecular pathogenetic mechanisms of PCOS, although it might contribute to the severity of hyperandrogenemia in women with PCOS and biochemical hyperandrogenism.

Association of the Pro12Ala polymorphism in peroxisome proliferator-activated receptor gamma2 with decreased basic metabolic rate in women with polycystic ovary syndrome.

OBJECTIVE: The peroxisome proliferator-activated receptor (PPAR)gamma is a transcription factor involved in glucose homeostasis and energy metabolism. A missense mutation at codon 12 in the PPARgamma2 has been associated with increased body mass index (BMI) and attenuated insulin resistance (IR) in polycystic ovary syndrome (PCOS). We have recently shown a decreased basic metabolic rate (BMR) in PCOS. The aim of the present study is to determine the prevalence of the Pro12Ala polymorphism of the PPARgamma2 gene and its associations with indices of IR and BMR in lean and slightly overweight PCOS women. DESIGN: Case-control association study involving 156 PCOS women with biochemical hyperandrogenism, chronic anovulation and polycystic ovarian morphology in ultrasound and 56 unrelated healthy controls. METHODS: Hormonal determinations were performed by electrochemiluminescence quantitation or RIA. BMR was measured by indirect calorimetry. All subjects were genotyped by a PCR-restriction fragment length polymorphism assay. RESULTS: Genotype frequencies of the Pro12Ala polymorphism in PPARgamma2 did not differ among PCOS women and control subjects. The presence of Pro12Ala polymorphism of PPARgamma2 was associated with lower BMR (P=0.04). This finding was valid in our subgroup of lean PCOS (BMI<25 kg/m(2)), in which the Ala variant was also associated with higher total testosterone values. CONCLUSION: The Pro12Ala polymorphism in the PPARgamma2 gene is associated with decreased BMR in women with PCOS and biochemical hyperandrogenemia. These young women are therefore at risk to increase their body weight and should restrict their energy intake by diet and enhance their energy expenditure by exercise.

The phosphodiesterase 5 inhibitor sildenafil stimulates angiogenesis through a protein kinase G/MAPK pathway.

cGMP-degrading pathways have received little attention in the context of angiogenesis. In the present study we set out to determine whether cGMP-specific phosphodiesterase 5 (PDE5) inhibition affects new blood vessel growth. Incubation of chicken chorioallantoic membranes (CAMs) in vivo with sildenafil increased vascular length in a dose-dependent manner. Moreover, incubation of cultured endothelial cells (ECs) with the PDE5 inhibitor promoted proliferation, migration, and organization into tube-like structures. The effects of sildenafil on the angiogenesis-related properties of EC could be blocked by pre-treatment with the soluble guanylyl cyclase (sGC) inhibitor ODQ or the protein kinase G (PKG) I inhibitor DT-3. In addition, over-expression of sGC in EC led to an enhanced growth and migratory response to sildenafil. To study the signaling pathways implicated in the sildenafil-stimulated angiogenic responses we determined the phosphorylation status of mitogen-activated protein kinase (MAPK) members. Incubation of cells with sildenafil increased both extracellular signal regulated kinase 1/2 (ERK1/2) and p38 phosphorylation in a time-dependent manner. Inhibition of MEK by PD98059 and p38 with SB203580 blocked sildenafil-induced proliferation and migration, respectively, suggesting that these MAPK members are downstream of PDE5 and mediate the angiogenic effects of sildenafil. PDE5 inhibitors could, thus, be used in disease states where neo-vessel growth is desired.

alpha 2 beta adrenoreceptor 301-303 deletion polymorphism in polycystic ovary syndrome.

alpha(2beta) adrenoreceptor 301-303 deletion polymorphism does not influence basal metabolic rate, insulin resistance or weight gain in Greek women with polycystic ovary syndrome.