Contribution of adenosine-producing ectoenzymes to the mechanisms underlying the mitigation of maternal-fetal conflicts.

The interactions taking place between mother and embryo have been the focus of detailed studies in recent years, where pregnancy is considered as an in vivo transplant. The immune systems of the mother and the embryo together establish a condition of tolerance, which lasts throughout the pregnancy. Alongside immunogenetic components, a contribution is provided by the ectoenzyme network, a chain of surface molecules mainly operating in closed environments and potentially providing inhibitory or activator signals. One of the soluble products of the ectoenzyme network with immunosuppressory potential is adenosine, a purine nucleoside that plays multiple roles in almost all tissues and organs. The hypothesis behind the work was studied in patients with recurrent pregnancy loss (RPL), an event which remains unexplained in over 50 percent of cases. To this aim, we analyzed the expression of CD39 (ectonucleoside triphosphate diphosphohydrolase 1, ENTPD1) and CD73 (ecto-5’-nucleotidase, NT5E), the main pathway for adenosine generation, in samples obtained from women with RPL. The study included the evaluation of the expression of TNF-alpha (a pro-inflammatory cytokine) and of an alternative pathway of adenosine generation run by CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) and PC-1 (ectonucleotide pyrophosphatase/phosphodiesterase 1, ENPP1). The results of this study highlight the existence of a network of surface enzymes expressed at the maternal/fetal interface and addressed to the production of adenosine. Perturbation of this network may induce a rescue pathway driven by CD38 and ENPP1. Ectoenzyme and inflammation may be considered now key elements in orchestrating the events leading to the interruption of pregnancy in the RPL sample analyzed and at the same potentially becoming therapeutic targets.

Effect of consumption of dark chocolate on oxidative stress in lipoproteins and platelets in women and in men.

The goal of this research was to investigate the effects of 3 weeks consumption of 50 g flavonoid-rich dark chocolate on lipoprotein oxidative stress in vitro and in vivo in 25 women compared to 25 men. Levels of thiobarbituric acid-reactive substances, conjugated dienes and hydroperoxide levels in HDL and LDL before and after consumption of dark chocolate were determined. Moreover in platelets of the same subjects NO and peroxynitrite levels were studied. TBARs concentration in women's HDL decreased by 26.7% while in men's HDL 23.4%; lipid hydroperoxides decreased in women's HDL by 62.8% while in men's HDL they decreased by 21.1%. Conjugate diene formation decreased in women's HDL by 55.9%, while in men's HDL it decreased by 49.2%. Moreover TBARs concentration decreased in women's LDL by 26.7% after supplementation and in men's LDL by 21.6%; lipid hydroperoxides decreased in women's LDL by 83.6% while in men's LDL they decreased by 64.7%. Moreover conjugate diene formation decreased in women's LDL by 48.2%, while in men's LDL it decreased by 21.6%. After supplementation peroxynitrite values decreased in women by 24% and in men by 18.6% while NO increased after supplementation by 15.7% compared to basal determination in women, and by 32.2% in men. This study showed that a short-term intake of dark chocolate might improve the lipoprotein profile in healthy humans, more so in women than in men, and this might exert a protective effect on the cardiovascular system.

Neurogenic potential of mesenchymal-like stem cells from human amniotic fluid: the influence of extracellular growth factors.

Amniotic fluids contain human stem cells, among which mesenchymal stem cells could be isolated. These cells have multipotent differentiation ability and no tumorigenic potential after transplantation in mice. These features make them good candidates for in vitro studies and for therapeutic purposes. The aim of this study was to isolate mesenchymal stem cell-like cultures from different amniotic fluids in order to study in vitro their neurogenic potential and assess if this process could be reproducible and standardized. We focused attention on the possible differential effects of soluble growth factors. Immunophenotypical and molecular characterization showed that the 31 amniotic fluid-derived cultures expressed mesenchymal markers as well as some stemness properties. These cells also appeared to be responsive to purines or acetylcholine showing an intracellular calcium increase, also reported for mesenchymal stem cells derived from other sources. Interestingly, in the presence of retinoic acid, these cells assumed a neuronal-like morphology. In addition, functional and molecular analyses revealed that retinoic acid-treated cells showed immature electric functional properties, the expression of neuronal markers and stemness genes. In conclusion, even if further investigations are required, the results presented here contribute to support the finding that amniotic fluid contains cells able to differentiate in vitro towards neural-like lineage in the presence of retinoic acid. The ability of retinoic acid to induce a possible neuronal progenitor culture makes the model useful to study a possible in vivo transplantation of these cells and to contribute to define the protocols for cell therapy.

[Identification of preterm labor: the role of the fibronectin and ultrasound cervicometry and their association]. / La identificazione del travaglio pretermine: ruolo della fibronectina e della cervicometria ecografica e loro associazione.

OBJECTIVE: The aim of this study was to evaluate the effectiveness of the fetal fibronetcin (fFN) test and ultrasonographic cervical length measurement used alone or in combination with each other in order to further improve the identification of patients in preterm labor. METHODS: Prospective multicenter observational study on patients between 24 and 32 weeks of gestation with symptoms of preterm labor (total patients = 132). The endpoint was the delivery at 34 weeks or more. The screening methods used were: the fFN test (group 1), the cervical length measurement by transvaginal ultrasound (group 2) or a combination of both tests (group 3) according to an established protocol. The statistical analysis was performed using the χ2 test using the SPSS software. RESULTS: Group 1: positive fFN test in 25.7% of cases, incidence of preterm birth (<34 weeks) of 18%. Group 2: cervical length <25 mm in 56.2% of cases, incidence of preterm birth (<34 weeks) of 18.5%. The negative predictive value is equivalent to 99.0% for the fFN test and 95.2% for cervicometry; combined use reaches 100%, compared to 54% positive predictive value. CONCLUSION: The identification of women at high risk of preterm delivery carried out with the fFN test or with transvaginal ultrasound cervicometry is clinically valid. The study also showed that the contextual use of biochemical and biophysical tests reaches a high negative predictive value (100%), making it a very useful method to identify patients truly at risk and to further reduce the incidence of non adequate treatment.

Expression patterns of two serine protease HtrA1 forms in human placentas complicated by preeclampsia with and without intrauterine growth restriction.

Preeclampsia (PE) and intrauterine growth restriction (IUGR) are pregnancy-specific disorders that have in common abnormal placental implantation, a marked proliferation of villous cytotrophoblastic cells and focal necrosis of the syncytiotrophoblast. Several studies show an ischemic placenta with a high-resistance vasculature, which cannot deliver an adequate blood supply to the feto-placental unit. The cause of PE is a matter of debate, but recently studies in mice suggest that the primary feto-placental lesions are sufficient to initiate the disease. HtrA1, a member of the family of HtrA proteins, is a secreted multidomain protein with serine protease activity. It is expressed in first and third trimester of gestation. In specimens from the first trimester of gestation, immunostaining for HtrA1 is generally found in both layers of villous trophoblast, syncytiotrophoblast and cytotrophoblast. Cytoplasm of extravillous trophoblast and extracellular matrix of cell islands and cell columns are labeled for HtrA1. Specimens from third trimester of gestation show a more intense positivity for HtrA1 in the syncytiotrophoblast than in cytotrophoblast. The extravillous trophoblast and the decidual cells, is positive for HtrA1. The purpose of this study is to investigate the expression pattern of HtrA1 in placentas from PE without IUGR (maternal PE) and with IUGR (fetal PE) by quantitative western blotting and immunohistochemistry. By quantitative western blotting analysis we observed a significant upregulation of approximately 30 kDa HtrA1 form in PE. Differently, we detected a significant total HtrA1 down-regulation in PE-IUGR. Moreover, immunostaining for HtrA1 was positive in the villous trophoblast, in the syncytial knots and irregularly in the fetal vessel walls in PE placentas while immunostaining for HtrA1was present particularly in the syncytial knots in PE-IUGR placentas. In conclusion, we suggest that the approximately 30 kDa HtrA1 form can be correlated to maternal PE while that the significant down-regulation of total HtrA1 can be correlated to placental PE. These HtrA1 alterations could be considered as possible markers to discriminate placental PE from maternal PE.

Nitric oxide and peroxynitrite platelet levels in women with small-for-gestational-age fetuses.

OBJECTIVE: The placenta produces reactive oxygen species (ROS) including nitric oxide (NO) and peroxynitrite (ONOO(-)) that have pronounced effects on placental function. Excessive ROS production may occur in pathological pregnancies, such as those complicated by small-for-gestational-age (SGA) fetuses. DESIGN: The aim of the present work was to study NO and ONOO(-) levels in platelets of pregnant women with SGA fetuses compared with a control group. SETTING AND POPULATION: The study was performed on 30 pregnant women with SGA fetuses (SGA group) and on 30 healthy pregnant women (appropriate-for-gestational-age [AGA] group) matched for maternal and gestational age. All women included in this study were in the third trimester of pregnancy. METHODS: Platelets were isolated by differential centrifugation. NO metabolites, after enzymatic conversion followed by the Griess reaction, were measured as nitrite by spectrophotometric detection. Peroxynitrite (ONOO(-)) levels were evaluated using the fluorescence probe 2,7-dichlorofluorescein diacetate (DCFDA). MAIN OUTCOME MEASURES: The following determinations were made: platelet nitric oxide and peroxynitrite levels in the SGA group and controls; inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS) and nitrotyrosine (N-Tyr) expression in the same groups. RESULTS: Our results show that both platelet NO and ONOO(-) levels were significantly higher in the SGA group than in the controls. CONCLUSION: Increased platelets levels of nitric oxide and peroxynitrite might play a role in the pathophysiology of intrauterine growth restriction. Further investigations are in progress to clarify if these molecules are pathogenetic factors, an epiphenomenon or a pathophysiological marker.

Potential role of culture mediums for successful isolation and neuronal differentiation of amniotic fluid stem cells.

In recent years, the use of stem cells has generated increasing interest in regenerative medicine and cancer therapies. The most potent stem cells derive from the inner cell mass during embryonic development and their use yields serious ethical and methodological problems. Recently, a number of reports suggests that another suitable source of multipotent stem cells may be the amniotic fluid. Amniotic fluid mesenchymal stem cells (AFMSCs) are capable of extensive self-renewal, able to differentiate in specialized cells representative of all three germ layers, do not show ethical restriction, and display minimal risks of teratomas and a very low immunogenity. For all these reasons, amniotic fluid appears as a promising alternative source for stem cell therapy. Their recent discovery implies a lack of knowledge of their specific features as well as the existence of a protocol universally recognized as the most suitable for their isolation, growth and long-term conservation. In this study, we isolated stem cells from six amniotic fluids; these cells were cultured with three different culture mediums (Mesenchymal Stem Cell Medium (MSCGM), PC-1 and RPMI-1640), characterized by cytofluorimetric analysis, and then either frozen or induced to neuronal differentiation. Even if the immunophenotype seemed not to be influenced by culture medium (all six samples cultured in the above-mentioned mediums expressed surface antigens commonly found on stem cells), cells showed different abilities to differentiate into neuron-like cells and to re-start the culture after short/long-term storage. Cells isolated and cultured in MSCGM showed the highest proliferation rate, and formed neuron-like cells when sub-plated with neuronal differentiation medium. Cells from PC-1, on the contrary, displayed an increased ability to re-start culture after short/long term storage. Finally, cells from RPMI-1640, even if expressing stem cells markers, were not able to differentiate in neuron-like cells. Further studies are still needed in order to assess the effective role of culture medium for a successful isolation, growth, differentiation and storage of AFMSCs, but our data underline the importance of finding a universally accepted protocol for the use of these cells.

Placental overexpression of transforming growth factor-beta3 in the HELLP syndrome.

OBJECTIVE: To evaluate the placental expression of transforming growth factor-beta3 (TGF-beta3) in patients with HELLP syndrome and pre-eclampsia compared to controls, and its correlation to Doppler velocimetry analysis of the utero-placental blood flow. STUDY DESIGN: Real-time PCR analysis was performed, after cesarean section, in placental samples from 10 women affected by HELLP syndrome, 10 women with pre-eclampsia and 10 controls. Pulsatility indices on Doppler waveform analysis from uterine and umbilical arteries were measured. RESULTS: The mean TGF-beta3 expression was significantly higher in patients with HELLP syndrome compared with the control group (p < 0.001), and no difference was observed in the pre-eclampsia group. TGF-beta3 expression correlated positively with umbilical PI (p < 0.001). CONCLUSIONS: TGF-beta3 may play a key role as regulator of a variety of cellular events occurring during HELLP syndrome, high local expression of this growth factor may be responsible for remodeling of the placental structure, which results in the dysfunction of maternal-fetal circulation.

HELLP syndrome and placental inflammatory pathology.

HELLP syndrome, acronym for hemolysis (H), elevated liver enzymes (EL), and low platelet count (LP), is a multisystemic disease that complicates pregnancy and is considered a severe variant of hypertensive disorders in pregnancy, that causes maternal and perinatal mortality and morbidity. The pathogenesis of HELLP syndrome is not completely understood and the obstetric approach with the induction of delivery is still the only specific therapy in HELLP syndrome. It is well known that the placenta and the incomplete trophoblast invasion of spiral arteries have a central role, but especially in severe pre-eclampsia and in the HELLP syndrome there is a systemic endothelial activation and damage. In this review we emphasize the inflammatory hypothesis and the role of inflammatory cytokines deriving from placenta in pre-eclampsia and HELLP syndrome, also in the light of our recent studies on cytokines pattern.

[Maternity leave and possible effects of working during pregnancy in 160 women]. / Il lavoro in gravidanza: studio dei possibili effetti del lavoro su 160 puerpere.

Three years from the approval of the law safeguarding pregnant workers and the foetus (D.Lgs. 151/01), awareness of these provisions and management of pregnancy in relation to their job were assessed in 160 pregnant workers using a questionnaire. Respondents were 147 subjects, mean age 29.4 +/- 4.6 years, employed as blue-collar workers (n=26); white-collar workers (n =47); health workers (n=16); school workers (n=14); workers in shops (n=6); policewomen (n =3) housewives (n=25). Moreprovisions and most of them took advantage of maternal leave. Pre-term birth (16%) and low birth weight (9%) were the mostfrequent events. There were no neonatal diseases. Blue-collar, white-collar work and work in the home were not associated with pre-term births or low birth weight. Analysis based on the fathers' job characteristics did not yield significant associations either. Although the small size of our sample does not allow to draw firm conclusions, it is interesting to note that more than 10% of respondents were not aware of the provisions protecting workers' pregnancy.

Sodium/potassium-adenosine triphosphatase on erythrocyte ghosts from pregnant women and its relationship to pregnancy-induced hypertension.

The regulation of electrolytes has been proposed as a possible determinant in hypertensive conditions, including pregnancy-induced hypertension. We report a study of sodium/potassium-adenosine triphosphatase in erythrocyte membranes of 59 patients with pregnancy-induced hypertension and 48 normotensive pregnant controls. The kinetics of the enzyme were also investigated, and the enzyme activity related to different degrees of pathology. A marked reduction of the enzyme activity was found in hypertensive patients, compared with controls. This reduction was greater when hypertension was associated with worse prognostic signs, such as proteinuria. These features are in agreement with the increased sodium content and the increased vascular reactivity found in pregnancy-induced hypertension. The enzyme activity appears to be decreased by means of a conformational modification of the enzyme sites, a phenomenon related to the hypertensive condition.

Altered lipid composition, increased lipid peroxidation, and altered fluidity of the membrane as evidence of platelet damage in preeclampsia.

OBJECTIVE: To assess lipid composition, lipid peroxidation, and fluidity of the membrane of platelets from preeclamptic women. METHODS: We studied 40 primigravid women at 28-32 weeks' gestation; 20 were preeclamptic and 20 were normotensive. After preparing platelet membranes, we extracted lipids, measured cholesterol and phospholipid concentrations, and calculated the proportion of unsaturated to saturated fatty acids. Lipid peroxides expressed as conjugated dienes were determined by spectrophotometry. Membrane fluidity was determined by means of fluorescent lipophilic probes. Statistical analysis was performed by the Student t test, with significance at P < .05. RESULTS: Cholesterol concentration, cholesterol-to-phospholipid ratio, the amount of unsaturated fatty acids, conjugated dienes, and membrane fluidity significantly increased in platelets from preeclamptic patients as compared with the normotensive women. CONCLUSIONS: The discrepancy between cholesterol increase and membrane fluidity increase is consistent with the increase in unsaturated fatty acid content. In the platelet membrane, unsaturated fatty acids constitute the larger substrate for lipid oxidation and can also take part in the formation of thromboxane. Therefore, platelet membrane damage in preeclampsia, through imbalance of thromboxane A2/prostacyclin production, may contribute to the onset or maintenance of vasoconstriction and hypertension.

Calcium and sodium transport in gestational hypertension.

The enzymatic activities of Na+/K+ ATPase and Ca2+ ATPase were determined on erythrocyte membranes from 9 normotensive and 9 gestational hypertensive pregnant women near term. A reduction in the activity of the Na+/K+ ATPase and a relative increase in the activity of the Ca2+ ATPase were found in the hypertensive patients, possibly due to a conformational alteration of erythrocyte membranes. This observation supports the possible role of the transmembrane cation transport in the pathogenesis of gestational hypertension.

Laparoscopic cryomyolysis: an alternative to myomectomy in women with symptomatic fibroids.

BACKGROUND: The purpose of this study was to determine whether cryomyolysis may present an alternative valid surgical procedure to hysterectomy or myomectomy for selected women with symptomatic fibroids who wish to preserve their uterus but do not desire future pregnancies. METHODS: Sixty-three women with symptomatic fibroids who refused either myomectomy or hysterectomy, requesting a conservative surgery for myomata, underwent laparoscopic cryomyolysis using a 3- to 5-mm or 8-mm cryoprobe (CRYOcare system). RESULTS: Our study showed a mean (+/-standard deviation) decrease of myoma volume of 60.3% (+/-20.7) and complete symptom relief in 83.6% (p < 0.001) of patients after a 12-month follow-up from cryomyolysis. No significant intra- or postoperative complications were noted. CONCLUSION: Cryomyolysis is an effective laparoscopic procedure for obtaining myoma shrinkage and symptom relief in women with symptomatic fibroids who desire to preserve their uterus.

Amniotic levels of vascular endothelial growth factor and nitric oxide at the second trimester in Down's syndrome.

OBJECTIVE: Since placentae in trisomy 21 show trophoblastic hypoplasia and hypovascularity, we investigated amniotic fluid vascular endothelial growth factor (VEGF) and nitric oxide (NO) in normal pregnancy and pregnancy complicated by trisomy 21. Furthermore, we investigated a possible role of NO in neurodegeneration of the brain in Down's syndrome. METHODS: We retrospectively assessed NO and VEGF on mid-trimester amniotic fluid from 15 women who had fetal Down's syndrome, and compared the results with those of 15 controls matched for age and gestation. RESULTS: In pregnancies complicated by trisomy 21, NO levels were significantly higher than in healthy controls (p < 0.001), whereas VEGF levels were significantly lower than in healthy controls (p < 0.05). CONCLUSIONS: Our results suggest that the high levels of NO and the low levels of VEGF observed in the amniotic fluid of fetuses with Down's syndrome may be a sign of an imbalance of placental vascularization and altered endothelial function. Overproduction of NO could contribute to pathological cell death in the central nervous system, a process that has been demonstrated in many neurodegenerative diseases.

Amniotic levels of nitric oxide in women with fetal intrauterine growth restriction.

OBJECTIVE: Many biochemical observations have shown that nitric oxide (NO) is involved in the vascular angiogenic activity of the fetoplacental unit. The aim of this study was to determine whether NO is implicated in the pathogenesis of intrauterine growth restriction (IUGR). METHODS: We retrospectively assessed amniotic fluid NO from second-trimester amniocentesis of 20 healthy normotensive women who subsequently developed IUGR and 20 controls. The same women were re-assessed at the third trimester when IUGR had developed and when the same 20 controls had shown normal pregnancy. Amniotic fluid NO was detected by discontinuous spectrophotometry and the Griess reaction. RESULTS: At the second trimester, NO levels in women with subsequent IUGR were significantly lower than in controls (4.1 +/- 0.2 microg/mg creatinine vs. 6.02 +/- 1.57 microg/mg creatinine, p < 0.001). At the third trimester, in women with IUGR, NO levels were significantly higher than in normal pregnancies (7.4 +/- 1.5 vs. 5.02 +/- 0.9 microg/mg creatinine, p < 0.001), and directly correlated with gestational age when growth restriction was diagnosed (r = 0.69, p < 0.001). CONCLUSIONS: Low levels of NO during the early second trimester may represent an impaired stimulus to vascular formation and endothelial regulation, inducing placental disease and subsequent fetal growth restriction. High levels of amniotic fluid NO during the third trimester may represent a compensation factor for maintaining adequate uteroplacental perfusion in pregnancies with IUGR.

Prediction of gestational hypertension or intrauterine fetal growth restriction by mid-trimester 24-h ambulatory blood pressure monitoring.

OBJECTIVES: To investigate clinical impact of 24-h ambulatory blood pressure monitoring (ABPM) on the prediction of hypertensive disorders of pregnancy and IUGR. METHODS: ABPM was performed in 334 normotensive non-proteinuric nulliparous women at 20 weeks' gestation. Arterial blood pressure patterns were analyzed by chronobiometry. RESULTS: Women who developed idiopathic IUGR (21) or PIH (33) showed a 24-h diastolic blood pressure mean significantly higher than the controls (69.2+/-1.8 mmHg and 73.5+/-6.2 vs. 62.2+/-1.5). Women with subsequent IUGR also showed a modification in BP rhythm. The most effective cut-off levels of 24-h diastolic blood pressure mean proved to be 67 for IUGR and 68 for hypertension. CONCLUSIONS: ABPM in the second trimester reliably predicts idiopathic IUGR and PIH. Both patients destined to develop gestational hypertension and those destined to develop IUGR show similar elevations in 24-h diastolic mean at 20 weeks' gestation.

Lymphocyte subset in HPV infection.

The outcome of HPV infection and its progression to neoplastic disease may be conditioned by the immune status of patients. In the present study we observed the systemic lymphocyte subset, with particular regard to NK cells and NK activity in 7 patients with HPV infection. The lymphocyte phenotypes in peripheral blood were studied using a panel of monoclonal antibodies, while NK activity was evaluated as percentage of lysis of K 562 target cells. We noticed a significant decrease of basal NK activity especially in patients with HPV 16-18 infection (p less than 0.001), without any difference in the absolute number of these cells.

Paget's disease of the vulva: advantages of demolitive surgery.

Four patients with vulvar Paget's disease were analysed retrospectively. In all cases the clinical manifestation was typical, with pruritus and visible lesion. None of the cases had a palpable cutaneous or subcutaneous mass but at the histologic exam a minimally invasive Paget's disease was detected. Treatment consisted of total vulvectomy and subsequent new reconstructive time; only one patient with clinically localized disease was treated with simple vulvectomy. Postoperative complications were minimal, thanks to the reconstructive time that assured a rapid recovery of the surgical injury and reconstruction of the urethral and vaginal meatuses. In all cases we obtained the complete removal of the lesion (negativity of resection margins), except in one case that was also the only one to have a recurrence after 40 months. The patients have been followed for an average of 58 months. Considering the results, we suggest demolitive surgery with subsequent reconstructive time in the treatment of vulvar Paget's disease.

Endometrioid carcinoma of the ovary. Retrospective study.

From 1985 to 1991, 9 patients with endometrioid carcinoma of the ovary were treated and followed at the University of Ancona, Department of Gynecology and Obstetrics. Four patients (44.4%) had Stage I disease, 1 (11.1%) Stage II, 1 (11.1%) Stage III and 3 (33.3%) Stage IV. Six patients (66.6%) had grade 2 of the disease and 3 (33.3%) grade 3. Two of the patients (22.2%) had synchronous endometrial carcinoma while 3 had histologic evidence of endometriosis at the time of presentation. All the patients received treatment of combination of surgery, polychemotherapy, hormone and/or immunotherapy. The overall survival rate after a median follow up of 26.6 months was 66.6%. A high survival (100%) was observed for patients with associated endometriosis.