Potential Role of Placental Klotho in the Pathogenesis of Preeclampsia.

The aim of this study was to analyze the placental expression and allele status of promoter region of Klotho in association with preeclampsia, which represents the most common hypertensive disease of pregnancy. Klotho mRNA and protein levels were determined using real-time PCR and Western blot, respectively, in placental tissue samples obtained from 34 patients affected with preeclampsia and 34 controls. A PCR-based genotyping analysis was carried out in the promoter region of Klotho gene. Moreover, expression levels of pluripotency markers, Nanog and Oct4, and telomere length were assessed using real-time PCR. Klotho mRNA and protein levels were reduced in preeclamptic placentas compared with controls. -744delA single-nucleotide polymorphism was significantly associated with preeclampsia. In pathological placentas, there was a downregulation of pluripotency markers and a reduced telomere length. This study is the first to evaluate the placental expression level of Klotho in association with preeclampsia. Further analyses will clarify its role in the pathogenesis of this pregnancy hypertensive disorder.

Number and size of uterine fibroids and obstetric outcomes.

OBJECTIVE: Estimating the impact of sonographically identified multiple or large (≥5 cm in diameter) fibroids on obstetric outcomes. METHODS: Retrospective cohort study of 219 women with uterine fibroids (identified on a routine second-trimester ultrasound survey over a 3-year period, 2010-2012) and their age-matched controls. Inclusion criteria were singleton pregnancy, delivery at >24 weeks of gestation and no pathological conditions (chronic hypertension, gestational diabetes or pre-existing diabetes mellitus, uterine anomalies or fetal malformations). RESULTS: Compared to women with no fibroids, women with multiple fibroids (n = 34) had a significantly higher rate of preterm birth (29.4% versus 5%, p < 0.001), cesarean section (73.5% versus 37%, p < 0.001) and breech presentation (11.8% versus 2.7%, p = 0.04). Women with large fibroids (n = 48) had a higher rate of preterm birth (16.7% versus 5%, p = 0.01) and pPROM (10.4% versus 0.5%, p < 0.001). By multivariate analysis, only multiple fibroids and previous preterm birth showed an independent significant association with preterm birth (OR = 7.37, 95% CI 2.50-21.68 and OR = 13.01, 95% CI 3.56-47.52, respectively). CONCLUSIONS: Women with uterine fibroids are at an increased risk of obstetric complications. In particular, multiple rather than large fibroids are associated with a significantly increased risk of preterm birth and cesarean delivery while large fibroids are associated with a higher risk of pPROM. No correlation with IUGR, placenta previa or placental abruption was found.

Oxytocin, its antagonist Atosiban, and preterm labor: a role for placental nitric oxide.

OBJECTIVE: The aim of the present study was to understand the role played by Atosiban, an oxytocin receptor antagonist, on trophoblastic human cells, and the molecular bases of its efficacy and safety in the treatment of preterm labor. NO, peroxinitrite production and NOS expression have been evaluated on placenta obtained from term and preterm labors. PATIENTS AND METHODS: We studied trophoblast cells isolated from selected placental tissue from 20 controls and 20 preterm patients after cesarean sections. Each sample was studied at basal state and after 2 hours incubation with oxytocin and Atosiban. RESULTS: Significant variations of NO levels, peroxynitrite production and iNOS and eNOS expression both in the preterm, term samples and in each of the considered groups were observed. In the control group Atosiban re-established NO levels that were reduced after incubation with oxytocin, while in preterm samples NO levels were not only re-established but, after incubation with Atosiban, significantly increased compared to basal levels. CONCLUSIONS: This confirms the beneficial role of Atosiban in prolonging the pregnancy of spontaneous labor at very early gestational periods. In conclusion, Atosiban might be an effective drug to prevent preterm labor, in the therapeutic approach to this pathology.

Coasting, embryo development and outcomes of blastocyst transfer: a case-control study.

This study compared the effect on blastocyst development and clinical outcome of coasting in women at increased risk of moderate-severe ovarian hyperstimulation syndrome (OHSS; n=389) with a control group matched for age and basal FSH that did not undergo coasting (n=386) in IVF/intracytoplasmic sperm injection (ICSI) cycles. The main outcome measures were rate of blastocyst development and live birth. More cycles progressed to the blastocyst stage in the coasted group (n=169) compared with the control group (n=83; 43.4% versus 21.5%; P<0.001). The biochemical pregnancy, clinical pregnancy and live birth rates were similar (46.5% versus 42.0%; 40.6% versus 37.8%; 31.6% versus 30.1%). The duration of coasting up to 4 days did not affect progression to blastocyst stage. The multivariate model showed that coasting (OR 1.73, P=0.004) and the number of oocytes retrieved (OR 1.17, P=0.001) were positively correlated with blastocyst formation. Coasting, a measure to reduce the risk of OHSS, does not impair blastocyst development or clinical outcome. Coasting should remain an effective measure to prevent OHSS.

Tranilast, an orally active antiallergic compound, inhibits extracellular matrix production in human uterine leiomyoma and myometrial cells.

OBJECTIVE: To determine the effect of tranilast (an antiallergic drug known to suppress fibrosis or to stabilize mast cells) on extracellular matrix production in human leiomyoma and myometrial cells. DESIGN: Laboratory study. SETTING: University-affiliated laboratory. PATIENT(S): Seven premenopausal women who were admitted to the hospital for myomectomy or hysterectomy. INTERVENTION(S): Cells were treated with tranilast (300 µM) for 48 hours to measure extracellular matrix and activin-A expression by real-time reverse-transcription polymerase chain reaction and/or immunocytochemistry. MAIN OUTCOME MEASURE(S): The expression of fibronectin, collagen1A1, versican, and activin-A in myometrial and leiomyoma cells. RESULT(S): Tranilast decreased fibronectin, collagen 1A1, and versican messenger RNA (mRNA) expression in human primary leiomyoma cell culture. Similar results were found in an immortalized human leiomyoma cell line. Tranilast also decreased the mRNA expression of fibronectin, collagen 1A1, and versican in human primary myometrial cells. The reduced expression of fibronectin and collagen 1 were observed by immunocytochemistry as well. Tranilast also reduced profibrotic growth factor, activin-A mRNA expression in primary myometrial and leiomyoma cells. CONCLUSION(S): Our results indicate that tranilast reduced fibronectin, collagen 1A1, versican, and activin-A expression in leiomyoma and myometrial cells, demonstrating its potential as an antifibrotic therapy for human leiomyomas.

Management of leiomyomas in perimenopausal women.

Uterine leiomyomas, commonly called fibroids, are the leading indication for hysterectomy in the United States. Incidence increases with age from menarche to perimenopause. Regardless of their generally benign neoplastic character, uterine fibroids are responsible for significant morbidity in a large proportion of women of reproductive age. As uterine leiomyomas generally regress after menopause, the general attitude when women are approaching perimenopausal age is to avoid treatment and wait for menopause and a spontaneous resolution. When it is decided that treatment is needed, the choice for peri- and postmenopausal women is often hysterectomy. In the present paper we point out aspects of leiomyoma management that are unique to the perimenopausal period, and address future directions in care. We conclude that the management of uterine leiomyomas should not be overlooked in the perimenopausal period merely on the grounds that the pathology and symptoms are unlikely to persist after the menopause; on the other hand, opting for a quick resolution with total surgical removal of the uterus, as seen at present in many cases, should be avoided. Studies on the impact of therapy for fibroids should be performed not exclusively with premenopausal women but also with perimenopausal and postmenopausal women, both users and non-users of hormone replacement therapy.

Role of activin-A and myostatin and their signaling pathway in human myometrial and leiomyoma cell function.

CONTEXT: Uterine leiomyomas are highly prevalent benign tumors of premenopausal women and the most common indication for hysterectomy. However, the exact etiology of this tumor is not fully understood. OBJECTIVE: The objective of the study was to evaluate the role of activin-A and myostatin and their signaling pathways in human myometrial and leiomyoma cells. DESIGN: This was a laboratory study. SETTING: Myometrial and leiomyoma cells (primary and cell lines) were cultured in vitro. PATIENTS: The study included premenopausal women who were admitted to the hospital for myomectomy or hysterectomy. INTERVENTIONS: Primary myometrial and leiomyoma cells and/or cell lines were treated with activin-A (4 nM) and myostatin (4 nM) for different days of interval (to measure proliferation rate) or 30 minutes (to measure signaling molecules) or 48 hours to measure proliferating markers, extracellular matrix mRNA, and/or protein expression by real-time PCR, Western blot, and/or immunocytochemistry. RESULTS: We found that activin-A and myostatin significantly reduce cell proliferation in primary myometrial cells but not in leiomyoma cells as measured by a CyQUANT cell proliferation assay kit. Reduced expression of proliferating cell nuclear antigen and Ki-67 were also observed in myometrial cells in response to activin-A and myostatin treatment. Activin-A also significantly increased mRNA expression of fibronectin, collagen1A1, and versican in primary leiomyoma cells. Finally, we found that activin-A and myostatin activate Smad-2/3 signaling but do not affect ERK or p38 signaling in both myometrial and leiomyoma cells. CONCLUSIONS: This study results suggest that activin-A and myostatin can exert antiproliferative and/or fibrotic effects on these cell types via Smad-2/3 signaling.

The impact of loop electrosurgical excision procedure (LEEP) for CIN 2,3 on spontaneous preterm delivery in twin pregnancies by assisted reproductive technique: preliminary data.

The objective of this study was to compare the frequency of spontaneous preterm delivery before 35 weeks in 7 dichorionic twin pregnancies obtained after loop electrosurgical excision procedure (LEEP) for cervical intraepithelial neoplasia (CIN) 2,3 with respect to 21 twin pregnancies without previous cervical treatment. All the pregnancies were obtained after assisted reproduction techniques (ART). Same age at delivery was observed between two groups (p = 0.81) and none of our twin pregnancies after LEEP had a threatened preterm labor while four controls (19%) underwent a spontaneous preterm delivery (p = 0.35). These preliminary data seem to indicate that LEEP may not be responsible of spontaneous preterm delivery in twin pregnancies subsequent to ART.

Early and late-onset pre-eclampsia.

There is discrepancy in the literature on the definitions of severe and early-onset pre-eclampsia. We aimed to determine those definitions for clinical purposes and to introduce them in the classification of the hypertensive disorders of pregnancy forpublication purposes. We circulated a questionnaire to the International Committee of the International Society for the Study of Hypertension in Pregnancy focusing on the thresholds for defining severe preeclampsia and the gestation at which to define early-onset preeclampsia, and on the definition and inclusion of the HELLPsyndrome or other clinical features in severe preeclampsia. The questions were closed, but all answers had space for more open detailed comments.There was a general agreement to define preeclampsia as severe if blood pressure was ⩾160mmHg systolic or 110mmHg diastolic. There was scarce agreement on the amount of proteinuria to define severity. The HELLP syndrome was considered a feature to include in the severe classification. Most investigators considered early-onset preeclampsia as that occurring before 34 weeks. A definition of pre-eclampsia is paramount for driving good clinical practice. Classifications on the other hand are useful to enable international comparisons of clinical data and outcomes. We used the results of this survey to update our previous classification for the purposes of providing clinical research definitions of severe and early onset pre- eclampsia that will hopefully be accepted in the international literature.

Prediction, medical illness and the risk of pre-eclampsia.

Preeclampsia, the leading cause of maternal and perinatal morbidity and mortality, has been recently considered not only a pregnancy disease but also a risk factor for developing diseases later in life. Preeclampsia is becoming a disease of interest to internists and not just obstetricians. Women who have had preeclampsia seem to be at higher risk of premature death, mortality from ischemic heart disease, cardiovascular diseases including ischemic heart disease and hypertension, fatal and non-fatal stroke, venous thromboembolism.renal failure, type 2 diabetes mellitus, hypothyroidism, and cognitive defects, although they appear surprisingly protected from cancer. Furthermore, having had preeclampsia isa problem not only for the mother's future health, but it also affects the offspring's adult health. Children born from preeclamptic pregnancies are more prone to hypertension, insulin resistance and diabetes mellitus, neurological problems, stroke, and mental disorders along their life.Whether preeclampsia is a risk factor for disease later in life or it creates long-term organ damage is an intriguing question. Understanding the etiological background may provide guidance for the prevention and follow-up of women who experience preeclampsia.

The effects of different concentrations of cocoa in the chocolate intaken by the mother on fetal heart rate.

OBJECTIVE: To analyze the effects of different concentrations (30% and 80%) of cocoa on fetal heart rate (FHR). STUDY DESIGN: One hundred pregnant women with uncomplicated gestation, matched for age and parity, underwent computerized FHR recording before and after the consumption of 30 g of 30% and 80% cocoa chocolate. After 1 week, those who had received 30% were shifted to 80% and vice versa to have a crossover. Computerized cardiotocography parameters (contractions, fetal movements, baseline FHR, accelerations greater than 15 bpm for 15 s, number of decelerations, minutes of high variability, short term variability in ms) were recorded and expressed as mean and SD. The differences were tested for statistical significance using the paired t test, with the significance at p < 0.05. The percent change after chocolate intake for accelerations and short-term FHR variation was calculated. RESULTS: The number of fetal movements, accelerations, the duration of episodes of high variation and the short-term FHR variation were significantly higher (p < 0.0001) after 80% cocoa intake. After 30% cocoa chocolate intake, only the number of accelerations was significantly increased. The percent change of the number of accelerations and the short-term FHR variation were significantly higher after 80% cocoa chocolate maternal intake. CONCLUSIONS: Maternal intake of dark chocolate has a stimulating action on fetal reactivity. The effect is more marked with high concentrations (80%) of cocoa. This finding is likely due to the pharmacological action of theobromine, a methilxanthine present in cocoa.

Uterine leiomyoma: available medical treatments and new possible therapeutic options.

CONTEXT: Uterine leiomyomas (fibroids or myomas) are benign tumors of the uterus and are clinically apparent in up to 25% of reproductive-age women. Heavy or abnormal uterine bleeding, pelvic pain or pressure, infertility, and recurrent pregnancy loss are generally associated with leiomyoma. Although surgical and radiological therapies are frequently used for the management of this tumor, medical therapies are considered the first-line treatment of leiomyoma. EVIDENCE ACQUISITION AND SYNTHESIS: A review was conducted of electronic and print data comprising both original and review articles on pathophysiology and medical treatments of uterine leiomyoma retrieved from the PubMed or Google Scholar database up to June 2012. These resources were integrated with the authors' knowledge of the field. CONCLUSION: To date, several pathogenetic factors such as genetic factors, epigenetic factors, estrogens, progesterone, growth factors, cytokines, chemokines, and extracellular matrix components have been implicated in leiomyoma development and growth. On the basis of current hypotheses, several medical therapies have been investigated. GnRH agonist has been approved by US Food and Drug Administration for reducing fibroid volume and related symptoms. In addition, the FDA also approved an intrauterine device, levonorgestrel-releasing intrauterine system (Mirena), for additional use to treat heavy menstrual bleeding in intrauterine device users only. Currently, mifepristone, asoprisnil, ulipristal acetate, and epigallocatechin gallate have been shown to be effective for fibroid regression and symptomatic improvement which are all in clinical trial. In addition, some synthetic and natural compounds as well as growth factor inhibitors are now under laboratory investigation, and they could serve as future therapeutic options.

PP008. Placental klotho gene in preeclampsia.

INTRODUCTION: An aging-suppressor gene, klotho, is a candidate factor for vascular disease because its deficiency leads to impaired endothelium-dependent vasodilation and impaired angiogenesis. This protein is involved in several metabolic pathways such as the insulin-like growth factor 1 (IGF-1), apoptosis, angiotensin-II-induced events in the kidney and oxidative stress. OBJECTIVES: The aim of this study was to determine the difference of klotho genotiping and expression in the placentas of women with normal and preeclamptic pregnancies. METHOD/DESIGN: Placental tissue was collected from normal pregnancies (n=12) and pregnancies complicated by Preeclampsia (n=12), matched for gestational age. Klotho genotyping and expression was determined using real-time quantitative polymerase chain reaction (PCR) and Western blot, respectively. RESULTS: A polymorphism for -744 G/A mutation was significantly more common in the pathological group, with an odds ratio (OR) of 3.00 (1.02-8.81; 95% CI). The expression levels of both klotho isoforms and of the short klotho isoform were lower (80%) in the Preeclampsia group as compared to matched controls. Results of Western Blot agreed with those from Real-Time PCR. CONCLUSION: In preeclamptic pregnancies there are a genotyping polymorphism and a reduced expression of klotho gene. Given its role in cardiovascular disease in aging, it may link preeclamptic mothers and their offsprings to long term cardiovascular outcomes.

How to read fetal heart rate tracings in labor: a comparison between ACOG and NICE guidelines.

The aim of this study was to assess reproducibility and clinical relevance of current guidelines on fetal heart rate interpretation in labor. Two obstetricians with comparable experience analyzed one hundred fetal heart rate tracings. One doctor made a first analysis using American College of Obstetricians and Gynecologists (ACOG) 2009 guideline's criteria; the other used National Institute for Health and Clinical Excellence (NICE) 2007 guideline's criteria; subsequently they repeated the evaluation crossing the guidelines used. The primary outcome of this experiment was to determine the time spent to evaluate the tracings, secondary outcomes were: the intraobserver concordance (concordance of the evaluation with the two systems for each investigator), the interobserver concordance (concordance between the interpretation given by each investigator) and. the concordance between operators' grading and actual outcome of labor. The interpretation of fetal heart rate tracings was longer using ACOG criteria. The intraobserver agreement was significant. The interobserver agreement was better using NICE guidelines. The same trend showed for the concordance between investigators' grading and actual outcomes There was more discordance in worse outcomes. Both guidelines are interesting and useful, but NICE seems easier to handle than ACOG.

Placental expression of CD100, CD72 and CD45 is dysregulated in human miscarriage.

CONTEXT AND OBJECTIVE: The etiology of miscarriage is often multifactorial. One major cause, immunological rejection of the fetus, has not been clearly elucidated. Our aim was to establish whether the semaphorin CD100, its natural receptor CD72, and the glycoprotein CD45, implicated in immune mechanisms, are involved in pregnancy loss by examining their placental expression with real-time PCR, immunohistochemistry and western blotting techniques. PATIENTS: Placenta tissue from 72 Caucasian women undergoing surgical uterine evacuation due to early spontaneous pregnancy loss between the 8(th) and 12(th) week of gestation was divided into four groups based on miscarriage number. Gestational age-matched placentas from 18 healthy women without a history of miscarriage undergoing voluntary pregnancy termination were the control group. Placenta from 6 Caesarean deliveries performed at 38-40 weeks of gestation was also studied. RESULTS: CD100, CD72 and CD45 were expressed in placenta and exhibited different mRNA and protein levels in normal pregnancy and miscarriage. In particular, protein levels were highly dysregulated around 10 weeks of gestation in first and second miscarriage placentas. The CD100 soluble form was produced and immediately shed from placental tissue in all samples. CONCLUSIONS: Fetal CD100, CD72 and CD45 seem to play a role in miscarriage. The present data support the involvement of the fetal immune system in pregnancy maintenance as well as failure.

Two successful pregnancies in a woman affected by cystic fibrosis: case report and review of the literature.

BACKGROUND: Pregnancies in patients with cystic fibrosis (CF) are subject to an increased risk of complications. METHODS: We have made a systematic review of the literature concerning pregnancies in women with CF to evaluate prognostic factors of pregnancy outcome such as lung function and nutritional status, also including and describing the case of a woman suffering from CF who had two pregnancies in her late thirties, both with a good outcome. RESULTS: Thirteen case series and 22 case reports involving 523 pregnancies in 401 women were extracted. 83.1% of 516 pregnancies whose outcome was known resulted in the delivery of live births, with preterm birth rate of 24%. Miscarriage occurred in 6.3% and therapeutic abortion in 10% of pregnancies. Indirect mother's death occurred in seven cases. In our case report, the course of the second pregnancy proved to be much more difficult than the first, with preterm delivery probably related to a worsening of lung disease in the third trimester of pregnancy. CONCLUSIONS: Pre-pregnant lung function as well as lung function deterioration, CF-related diabetes mellitus and weight gain in pregnancy, are parameters to consider in the counseling about the outcome of pregnancy.

Glutamate in vitro effects on human term placental mitochondria.

OBJECTIVE: Oxidative stress may affect the functionality of placental mitochondria, thus contributing to serious complications. For this reason research of protective substances is of great importance. Our aim was to evaluate, in mitochondria isolated from human term placentas, the effect of in vitro glutamate supplementation on their susceptibility to oxidation, on the chemico-physical characteristics of mitochondrial membranes, and on peroxidase and nitric oxide synthase (NOS) activities. METHODS: The study was performed on mitochondria isolated from 20 healthy human term placentas. Specific exclusion criteria were: conception by assisted reproduction, chromosomal or other fetal, uterine or placental anomalies, gestational diabetes, preeclampsia, intrauterine growth restriction (IUGR), a history of smoking and hypertension, proteinuria, renal, cardiovascular, hepatic, and endocrine disease, metabolic disorders, and current infection or history of all types of infection. RESULTS: Incubation with glutamate determined a reduced susceptibility to oxidative stress, an increase in mitochondrial membrane fluidity, and a decrease of both peroxidase and NOS activities. CONCLUSIONS: On the basis of the observed results, we can hypothesize a role for glutamate in the control of lipid peroxidation extent in physiological pregnancies, as well as in the prevention of free radical-linked complications that can affect the health of both mother and fetus.

Topical Imiquimod 5% cream therapy for external anogenital warts in pregnant women: report of four cases and review of the literature.

OBJECTIVES: Imiquimod 5% cream (Aldara, Meda Farm, Milan, Italy) is largely used for the treatment of anogenital warts. However, its use during pregnancy is not consolidated with only a small number of patients worldwide that have been treated. The aim of this study is to assess the first line therapeutic efficacy and safety of Imiquimod 5% cream in pregnant women with external anogenital warts, including extensive condylomata. STUDY DESIGN: Four pregnant women's with external anogenital warts were treated with Imiquimod 5% cream, three times a week for four weeks. RESULTS: Mean number of warts treated per patient was 4.5 (5 SD) and mean area treated was 3.2 cm(2) (1.7 SD). A complete response was observed in two women, while two women had a partial response with a rate of clearing of 70% and 84% respectively. No cases of severe adverse local effects were observed in the sites of Imiquimod application. No adverse fetal outcomes or fetal and neonatal abnormalities were observed. No complications were observed in the postpartum and follow-up period. CONCLUSIONS: Anogenital warts treatment with Imiquimod in pregnancy seems to be promising and not compromise a good pregnancy outcome, in extensive condylomata too. These preliminary data need to be confirmed by larger studies. Similarly, no definitive conclusion may be obtained form a systematic review of the English literature.

Coenzyme Q10 content in follicular fluid and its relationship with oocyte fertilization and embryo grading.

BACKGROUND: No data are available on the presence and content of Coenzyme Q10 (CoQ10) in human follicular fluid and its role. OBJECTIVE: To assess the presence and concentration of CoQ10 in human follicular fluid in relation to oocyte fertilization. METHODS: CQ10 content was measured in follicular fluid obtained from 20 infertile women undergoing ovarian stimulation program for in vitro fertilization. CoQ10 levels were assayed by high-performance liquid chromatography system and normalized for follicular cholesterol and protein levels. Oocyte morphology and embryo grading were assessed. RESULTS: CoQ10/Protein levels resulted significantly in mature versus dysmorphic oocytes. Similarly, CoQ10/Cholesterol was significantly higher in grading I-II versus grading III-IV embryos. CONCLUSIONS: This study is the first demonstration of the presence of CoQ10 in the human follicular fluid. Although the biological and endocrine mechanism of CoQ10 in the follicular fluid and its correlation with oocyte and embryo development is unclear, a new step may be the administration of CoQ10 in infertile women to evaluate the biological and reproductive outcomes.

Plasma coenzyme Q10 is increased during gestational diabetes.

OBJECTIVES: To determine plasma CoQ(10) concentration in the course of gestational diabetes mellitus. STUDY DESIGN: The assessment was provided longitudinally during the third trimester of pregnancy in 40 women with gestational diabetes mellitus (GDM) and 40 normal controls. CoQ(10) was measured with the HPLC method. CoQ(10) results were also normalized to plasma cholesterol concentration (nmoles/mmoles). Plasma samples were collected longitudinally throughout the third trimester. RESULTS: No statistically significant difference of plasma CoQ(10)/cholesterol levels between GDM patients and controls at 28-32 and 32-36 weeks of gestation, this difference was significant in late pregnancy (36-40 weeks), similarly, in the same gestational period, there was an increased level of HOMA-IR as index of insulin resistance ORAC as index of oxidative stress. CONCLUSIONS: Since coenzyme Q(10) is believed to be an important cellular antioxidant defence, higher levels of CoQ(10) in GDM patients may be a compensatory mechanism, in response to an activated oxidative stress, probably associated to hyperglycaemia and insulin resistance.