Pro-inflammatory cytokine alterations in recent onset anorexia nervosa adolescent female patients before and after 6 months of integrated therapy: A case-control study.

Anorexia nervosa (AN) is a complex disorder affecting mainly, but not only, teenagers. Researchers agree that AN is deeply associated with a pro-inflammatory state following an impaired immune system, resulting from altered levels of cytokines such as IL-1ß and TNF-α, also impacted by the frequent depressive states. Thus, this case-control study aimed to evaluate the relationship between patients suffering from AN undergoing specialized eating disorder treatment for AN and pro-inflammatory cytokines. To reach our purpose, we assessed eating-related psychopathology and depressive symptoms and measured serum concentration of pro-inflammatory cytokines IL-1ß, IL-6, IL-8, and TNF-α before and after 6 months of integrated therapy (which included psychopharmacotherapy, psychotherapy, and nutritional treatment), to define whether selected pro-inflammatory cytokines could be considered a pathophysiological marker of the disorder. A sample of 16 young female patients with early diagnosis of AN, and without any previous treatment, and 22 healthy controls matched by age, sex, and socioeconomic status were enrolled. After 6 months of integrated therapy, a significant decrease of all selected pro-inflammatory cytokines was detected. In addition, an improvement in the anxiety-depressant aspects was also noted. In conclusion, the results obtained suggest that pro-inflammatory cytokines are indeed related to the pathophysiology of AN. However, further investigations, involving larger samples of patients with distinct subtypes of AN, are essential to confirm the current findings.

Evaluation of antioxidative and diabetes-preventive properties of an ancient grain, KAMUT® khorasan wheat, in healthy volunteers.

PURPOSE: Recently, there was an increasing interest on the use of ancient grains because of their better health-related composition. The aim of this study was to evaluate in healthy human subjects the antioxidative and diabetes-preventive properties of ancient KAMUT® khorasan wheat compared to modern wheat. METHODS: The study was a randomized, non-blind, parallel arm study where the biochemical parameters of volunteers with a diet based on organic whole grain KAMUT® khorasan products, as the only source of cereal products were compared to a similar replacement diet based on organic whole grain modern durum wheat products. A total of 30 healthy volunteers were recruited and the intervention period lasted 16 weeks. Blood analyses were performed before and after the diet intervention. The effect of KAMUT® khorasan products on biochemical parameters was analyzed by multiple quantile regression adjusted for age, sex, physical activity and BMI compared to data at baseline. RESULTS: Subjects receiving KAMUT® khorasan products showed a significantly greater decrease of fat mass (b = 3.7%; CI 1.6-5.5; p = 0.042), insulin (b = 2.4 µU/ml; CI 0.2-4.2; p = 0.036) and a significant increase of DHA (b = - 0.52%; CI - 1.1 to - 0.12; p = 0.021). CONCLUSIONS: Our study provides evidence that a substitution diet with KAMUT® khorasan wheat products can reduce some markers associated to the development of type-2 diabetes compared to a diet of modern wheat.

Effect of 1-y oral supplementation with vitaminized olive oil on platelets from healthy postmenopausal women.

OBJECTIVE: Olive oil is the main fat source in the Mediterranean diet and shows a protective role against aging and related diseases. Osteoporosis represents a serious health problem worldwide and is associated with an increased risk for fractures and mortality. Nutrition should be part of bone disease prevention strategies, especially in light of the aging population and the effect of diet on bone health. The aim of this study was to investigate whether oral supplementation with extra virgin olive oil (VOO) enriched with vitamins D3, K1, and B6 (VitVOO) is able to modify some physicochemical and functional plasma membrane properties and nitrosative stress markers status. METHODS: In this single-center, randomized placebo-controlled trial, 60 postmenopausal women were administered either VitVOO or placebo (PlaVOO). After 1 y of oral supplementation, platelet membrane fluidity changes, Na+/K+-ATPase activity, serum nitric oxide, and peroxynitrite levels were determined in participants. RESULTS: After 1 y (time 1), women taking VitVOO showed lower nitric oxide levels than those taking PlaVOO; the same trend was found for peroxynitrite levels. As far as membrane fluidity was concerned, a significant decrease in anisotropy of diphenylhexatriene and trimethylammonium-diphenylhexatriene at time 1 in VitVOO participants compared with PlaVOO was found. Finally, Na+/K+-ATPase activity showed a significant increase after VitVOO supplementation. CONCLUSION: The supplementation of VitVOO into the diet of postmenopausal women could represent a proper tool for platelet function and a useful strategy against nitrosative stress and related diseases, thus confirming the antioxidant role played by the added vitamins.

Platelet-Derived NO in Subjects Affected by Type 2 Diabetes with and without Complications: Is there any Relationship with their Offspring?

Macro- and microvascular complications are currently the principal causes of morbidity and mortality in patients with diabetes mellitus. Aim of this study was to determine if type 2 diabetic patients with nephropathy and coronary artery disease showed altered platelet-derived nitric oxide (NO) production, compared with diabetic subjects without complications, and if this alteration is also present in their diabetic offspring. In this case-control observational study, platelet NO and peroxynitrite content was determined on plasma from 60 male adult type 2 diabetic patients and 60 male offspring type 2 diabetic patients. Plasmatic levels of homocysteine were also determined in the same individuals. Moreover, Western blot analysis of platelet lysates was performed with specific monoclonal antibody for endothelial (eNOS) and inducible (iNOS) nitric oxide synthase. Our study showed a lower piastrinic production of NO in the group of parents without complications (ADH), compared with the group of offspring without complications (YDH) and with the groups of parents with complications. Furthermore, we observed a lower synthesis of peroxynitrite in platelets from the ADH group than in the groups of patients with complications, and in the YDH group compared with all other groups. Subjects from YDH group also showed lower iNOS expression, compared with all other groups. Our data suggest that alterations in nitric oxide metabolism may represent potential risk factors in type 2 diabetes complications, such as nephropathy and cardiovascular diseases, leading to development of new therapeutic strategies in order to delay and prevent the onset of such complications.

Placental expression of endothelial and inducible nitric oxide synthase and NO metabolism in gestational hypertension: a case-control study.

OBJECTIVE: Hypertension is one of the most common medical disorders in pregnancy and a role of nitric oxide (NO) metabolism has been described. Thus, the present work aimed at determining placental gene expression of eNOS and iNOS, to measure NO and ONOO(-) levels in patients with gestational hypertension (GH). METHODS: Fifteen patients with GH and 15 healthy pregnant controls were enrolled in the study. Placental tissue was taken immediately after delivery and was stored at -80 °C until analysis. A piece of frozen tissue was homogenized in the appropriate buffer. Total RNA was extracted and was reverse transcribed to obtain complementary DNA that was used for real-time PCR for iNOS and eNOS expression, whereas NO and ONOO(-) production were measured by commercially available kits. RESULTS: Placental eNOS and iNOS mRNA levels were significantly reduced in GH when compared to controls. NO and ONOO(-) production were both significantly higher in GH than controls. CONCLUSIONS: The reduced eNOS and iNOS gene expression in women with GH reinforces the hypothesis that the mechanisms involving NO pathways, may promote oxidative damage, by contributing to the reduced blood flow and increased resistance in the feto-maternal circulation and suggests the use of NO modulators as useful tools in GH management.

Higher Levels of Oxidized Low Density Lipoproteins in Alzheimer's Disease Patients: Roles for Platelet Activating Factor Acetyl Hydrolase and Paraoxonase-1.

Alzheimer's disease (AD) is associated with oxidative damage of low density lipoproteins (ox-LDL). In order to investigate whether higher levels of ox-LDL are related to alterations of the activity of enzymes involved in lipid metabolism, we studied the activity of paraoxonase-1 (PON1) and platelet activating factor acetylhydrolase (PAF-AH) in AD patients and the relationship between biochemical markers and severity of the disease. Levels of ox-LDL, PON1 (paraoxonase, arylesterase, and lactonase activities), and PAF-AH activity were evaluated in plasma from 49 patients affected by AD and from 34 control subjects matched for gender and age. Our results demonstrated alterations in the activities of PON1 and PAF-AH in AD patients compared to controls and showed, for the first time, a relationship between the activities of these enzymes, ox-LDL levels, and severity of the disease. A significant negative correlation was observed between the ratio PON1/PAF-AH and ox-LDL. Whatever the causes that contribute to a systemic oxidative stress in AD, our results have shown that AD patients exhibit higher PAF-AH activity than control subjects and higher ox-LDL. This phenomenon, in combination with diminished PON1 in these patients and, consequently, the relatively lower ratio PON1/PAF-AH activity, could contribute to inflammation and oxidative stress of plasma lipoproteins.

Effect of 1-year dietary supplementation with vitaminized olive oil on markers of bone turnover and oxidative stress in healthy post-menopausal women.

Osteoporosis represents a serious health problem worldwide associated with an increased risk of fractures and mortality. Nutrition should form part of bone disease prevention strategies, especially in the light of the population ageing and the diet effect on bone health. Thus the study aimed at verifying whether 1 year of oral supplementation with either extra virgin olive oil (VOO) enriched with vitamins D3, K1 and B6 (VitVOO) or VOO used as placebo (PlaVOO) is able to modify some bone turnover and oxidative stress markers. Bone mineral density (BMD) was assessed in 60 healthy post-menopausal women together with the bone vitamin K status by measuring undercarboxylated osteocalcine (ucOC) plasma levels, the ratio between ucOC and carboxylated osteocalcine (UCR) and the relations with oxidative stress markers. After 1 year (T 1), subjects taking VitVOO showed lower ucOC levels than those taking PlaVOO; the same trend was found for UCR. As far as BMD is concerned, a significant increase in T-score at T 1 in VitVOO subjects compared to PlaVOO was found. All oxidative stress markers as thiobarbituric acid reactive substances, lipid hydroperoxides and conjugated dienes showed a significant reduction after VitVOO supplementation, whilst plasma total antioxidant capacity values was significantly increased in VitVOO group compared to PlaVOO group at T 1. It might be suggested that the use of VitVOO in the diet of post-menopausal women could represent a proper tool for bone protection and a useful strategy against oxidative stress and related diseases, thus confirming the antioxidant role played by the added vitamins.

Effects of in vitro supplementation with Syzygium cumini (L.) on platelets from subjects affected by diabetes mellitus.

The aim of this study was to assess the in vitro effects of Syzygium cumini (L.) (Sc) incubation on platelets from patients with diabetes, in order to test its efficacy as a potential adjuvant therapy. This study was performed on 77 patients with diabetes [29 in good (DMgc) and 48 in poor glycemic control (DMpc)] and 85 controls. In patients, platelets were analyzed at recruitment and after in vitro Sc incubation (final concentration of 200 µg/ml for 3 hours at 37 °C), whereas in controls only basal evaluation was performed. Lipoperoxide and nitric oxide (NO) levels, superoxide dismutase (SOD) and Na(+)/K(+) ATPase activities, total antioxidant capacity (TAC), and membrane fluidity tested by anisotropy of fluorescent probes 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 1-6-phenyl-1,3,5-hexatriene (DPH) were determined. Collagen-induced platelet aggregation was also evaluated. In vitro Sc activity counteracts oxidative damage, by improving platelet function through augmented membrane fluidity and Na(+)/K(+) ATPase activity; it also enhances antioxidant system functionality by increasing NO levels, SOD activity, and TAC and by decreasing lipoperoxide levels both in whole samples and in DMgc and DMpc. In addition, a slight tendency towards collagen-induced platelet aggregation decrease after Sc was observed. However, all these parameters, even after improvement, did not reach the levels of control subjects. Our results suggest that Sc may have a preventive and protective effect in oxidative damage progression associated with diabetes mellitus and its complications. If our data will be confirmed, Sc supplementation might become a further tool in the management of this disease, especially in view of its easy availability, safety, low cost, and absence of side effects.

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.

Oxidative Stress and Erythrocyte Membrane Alterations in Children with Autism: Correlation with Clinical Features.

It has been suggested that oxidative stress may play a role in the pathogenesis of Autism Spectrum Disorders (ASD), but the literature reports somewhat contradictory results. To further investigate the issue, we evaluated a high number of peripheral oxidative stress parameters, and some related issues such as erythrocyte membrane functional features and lipid composition. Twenty-one autistic children (Au) aged 5 to 12 years, were gender and age-matched with 20 typically developing children (TD). Erythrocyte thiobarbituric acid reactive substances, urinary isoprostane and hexanoyl-lysine adduct levels were elevated in Au, thus confirming the occurrence of an imbalance of the redox status of Au, whilst other oxidative stress markers or associated parameters (urinary 8-oxo-dG, plasma radical absorbance capacity and carbonyl groups, erythrocyte superoxide dismutase and catalase activities) were unchanged. A very significant reduction of Na(+)/K(+)-ATPase activity (-66%, p<0.0001), a reduction of erythrocyte membrane fluidity and alteration in erythrocyte fatty acid membrane profile (increase in monounsaturated fatty acids, decrease in EPA and DHA-ω3 with a consequent increase in ω6/ω3 ratio) were found in Au compared to TD, without change in membrane sialic acid content. Some Au clinical features appear to be correlated with these findings; in particular, hyperactivity score appears to be related with some parameters of the lipidomic profile and membrane fluidity. Oxidative stress and erythrocyte membrane alterations may play a role in the pathogenesis of ASD and prompt the development of palliative therapeutic protocols. Moreover, the marked decrease in NKA could be potentially utilized as a peripheral biomarker of ASD.

Alzheimer's disease and diabetes: new insights and unifying therapies.

Several research groups have begun to associate the Alzheimer Disease (AD) to Diabetes Mellitus (DM), obesity and cardiovascular disease. This relationship is so close that some authors have defined Alzheimer Disease as Type 3 Diabetes. Numerous studies have shown that people with type 2 diabetes have twice the incidence of sporadic AD. Insulin deficiency or insulin resistance facilitates cerebral ß-amyloidogenesis in murine model of AD, accompanied by a significant elevation in APP (Amyloid Precursor Protein) and BACE1 (ß-site APP Cleaving Enzime 1). Similarly, deposits of Aß produce a loss of neuronal surface insulin receptors and directly interfere with the insulin signaling pathway. Furthermore, as it is well known, these disorders are both associated to an increased cardiovascular risk and an altered cholesterol metabolism, so we have analyzed several therapies which recently have been suggested as a remedy to treat together AD and DM. The aim of the present review is to better understand the strengths and drawbacks of these therapies.

Impact of gender on platelet membrane functions of Alzheimer's disease patients.

There are many evidences suggesting that oxidative stress is one of the earliest events in Alzheimer disease (AD) pathogenesis and plays a key role in the development of the AD pathology. The existence of substantial gender-related differences in the clinical features of AD has been recently confirmed (i.e. pathophysiologic features and epidemiologic trends). In addition, study results appear to indicate that the etiopathogenetic mechanisms of AD differ significantly in the 2 sexes. Based on previous results regarding changes in AD platelet plasma membrane, the purpose of the present study was to assess the impact of gender in the same model above reported. In particular we aimed at studying platelets from AD patients (M-AD and F-AD) and matched controls (M-C and F-C), divided into gender, by studying nitric oxide (NO) and peroxynitrite (ONOO(-)) production, the intracellular Ca(2+) concentration ([Ca(2+)]i), membrane Na(+)/K(+)-ATPase activity and fluidity. NO production was significantly elevated in platelets from both F-AD and M-AD compared to matched controls. M-AD showed NO production significantly higher than F-AD and it was the same between M-C and F-C. A similar trend was seen for ONOO(-). Platelets of both M-AD and F-AD had intracellular Ca(2+) concentrations significantly higher than F-C and M-C, while membrane Na(+)/K(+)-ATPase activity showed an opposite trend, but these differences are still significant. M-AD male subjects showed a significantly increased DPH fluorescence anisotropy (r) compared with controls, while for F-AD this discrepancy was not significant. The difference in DHP fluorescence anisotropy remained significant between M-AD and F-AD as well as between M-C and F-C. The TMA-DPH fluorescence anisotropy showed the same trend, but there were no significant differences between M-AD and F-AD, as well as between controls. The results of the current research support the conclusion that F-AD is not at greater risk than M-AD for oxidative stress injuries. Studies on gender differences could lead to a higher probability of improved health outcomes via better-targeted therapies.

Environmental and genetical aspects of the link between pregnancy, birth size, and type 2 diabetes.

Exposure of the fetus to the intrauterine milieu can have profound effects on the health of the offspring in adulthood. These observations are highly reproducible in many populations worldwide although the mechanisms behind them remain elusive. The 'thrifty phenotype' hypothesis proposes that poor fetal nutrition leads to programming of metabolism and an adult phenotype that is adapted to poor but not plentiful nutrition. Results of a series of studies demonstrate the powerful influence of the mother's metabolic state on whether the emerging adult develops obesity and hyperinsulinemia. Importantly, these attributes can be passed on to the next generation non-genetically and can be reversed and prevented. Such hypothesis has been expanded on by the "Developmental Origins of Health and Disease" (DOHaD) hypothesis which describes the origin of adult disease in terms of fetal developmental 'plasticity' or the ability of the fetus to respond to poor in-utero conditions. A wealth of epidemiological evidence has provided a convincing link between a sub-optimal gestational environment and an increased propensity to develop adult onset metabolic disease. In this paper the factors that participate in the programming of the fetus and infants that lead to endocrine dysfunction in postnatal life are reviewed.

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.

Nitric oxide and peroxynitrite platelet levels in gestational hypertension and preeclampsia.

The aim of the study was to investigate platelet nitric oxide (NO) pathways in women with Gestational Hypertension (GH), Preeclampsia (PE) and Controls. Platelet NO(x) and peroxynitrite (ONOO(-)) levels, inducible (iNOS) and endothelial nitric oxide synthase (eNOS) and Nitrotyrosine expression (N-Tyr) in 30 women with GH, 30 with PE and 30 healthy pregnant controls, age, parity and gestational age-matched, were assessed. Platelet NO(x) and ONOO(-) levels were significantly higher in GH and PE vs. Controls, with higher levels in GH vs. PE. At the same way, iNOS and N-Tyr were significantly higher in GH and PE vs. Controls, with higher levels in GH vs. PE. Since GH expressed higher amount of NO metabolites and higher activation of iNOS compared to PE, we can hypothesize that the severity of hypertensive pathology is almost not related to only NO metabolism, this research confirmed that GH and PE are associated with marked changes in NO pathways; it is not easy to understand if they could be interpreted as causes or consequence of these pathologic states.

Oxidative stress in ischaemic stroke.

BACKGROUND: Production of reactive oxygen species after ischaemic stroke may enhance tissue damage through multiple molecular pathways. MATERIALS AND METHODS: In this study, we examined the serum levels of lipoperoxide and hydroperoxide, conjugated dienes and total antioxidant capacity levels in 50 patients with acute ischaemic stroke (T0) to evaluate the possibility to use them as specific biochemical markers for cerebral ischaemia. Determinations were repeated after a month (T1) to correlate their relative changes with clinical evolution. RESULTS: Lipoperoxide, hydroperoxide and conjugated diene levels in platelets were significantly higher in the early stages with respect to their late evaluation. On the contrary, total antioxidant capacity showed a significant increase at T1 with respect to T0. A significant negative correlation between total antioxidant capacity and NIHSS score at T0 and T1 was found. There was a significant positive correlation between lipoperoxide, hydroperoxide and conjugated dienes levels and NIHSS score at T0 and at T1. CONCLUSIONS: These findings suggest that changes in free radical generation and consequent oxidative stress may have a role in the pathogenesis of acute ischaemic lesions. The activation of defence mechanisms like total antioxidant capacity could be involved in the limitation of ischaemic damage progression.

Alterations of platelet biochemical and functional properties in newly diagnosed type 1 diabetes: a role in cardiovascular risk?

BACKGROUND: The involvement of platelets in the pathogenesis of diabetic vascular complications is supported by several studies. Type 1 diabetic (T1D) platelets show increased adhesiveness and aggregation related to a modification of nitric oxide synthase activity. Moreover, different cell types from diabetic patients showed a decreased membrane Na(+) /K(+) -ATPase activity, which might be involved in diabetic complications. The aim of this study was to investigate whether T1D at onset is able to induce alterations of platelet physicochemical and functional properties and whether these changes are affected by hyperglycaemia. METHODS: The study was performed on 50 young subjects: 30 patients (1-14 years) affected by T1D and 20 age- and gender-matched healthy subjects. We analyzed platelet membrane fluidity by fluorescent anisotropy of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene and 1,6-diphenyl-1,3,5-hexatriene, Na(+) /K(+) -ATPase, nitric oxide, and peroxynitrite production. RESULTS: In T1D subjects, we observed an increased fluidity in the plasma membrane outer part and greater rigidity in the internal part compared with that in controls. Na(+) /K(+) -ATPase activity and nitric oxide levels were significantly reduced, while peroxynitrite production was increased compared with that in controls. Moreover, correlations found between the above parameters were correlated with fasting glycaemia and haemoglobin A(1c). CONCLUSIONS: T1D patients exhibit structural and functional modifications of platelet membrane properties and alterations of nitric oxide metabolism due to diabetes per se and not to chronic hyperglycaemia, insulin therapy, or ageing. These results support the hypothesis that oxidative attack could be an important early event in the pathogenesis of diabetic complications.

Anorexia nervosa: a role for L-arginine supplementation in cardiovascular risk factors?

OBJECTIVE: The aim of the present study was to evaluate the effects of a short-term oral L-arginine treatment on platelet NO production, intracellular calcium concentration, iNOS and eNOS expression, in AN patients. METHOD: Forty outpatients belonging to restricting subtype and 40 normal participants age and sex matched were enrolled in the study. RESULTS: NO production was significantly elevated in the platelets from AN patients compared with controls while [Ca(2+)](i) was significantly decreased in patients with respect to controls. Western blot analysis demonstrated that iNOS isoform was more pronounced in the cell lysates from AN patients than controls. After supplementation with L-arginine, both NO production and [Ca(2+)](i) seem to return to control levels, suggesting a probable recovery of their metabolisms. The same was found after western blot analysis of NOS expression. DISCUSSION: The results here proposed can be considered highly indicative of a positive effect of L-arginine supplementation on platelet NO production in AN patients.

Nitric oxide platelet production in spontaneous miscarriage in the first trimester.

OBJECTIVE: To investigate the role played by platelet nitric oxide (NO) metabolism in patients with spontaneous miscarriage (SM) and recurrent spontaneous miscarriage (RSM) compared with healthy pregnant women. DESIGN: Retrospective case-control study. SETTING: Patients and controls in an academic research environment. INTERVENTION(S): None. PATIENT(S): Thirty singleton pregnant women who experienced SM, nine singleton pregnant women who presented with RSM, and 30 singleton healthy pregnant women matched for age, parity, and gestational age were enrolled. MAIN OUTCOME MEASURE(S): NO levels and peroxynitrite (ONOO(-)) production; moreover, inducible NO synthase (iNOS), endothelial NO synthase (eNOS), and nitrotyrosine expression (N-Tyr) were observed in the same samples. RESULT(S): A significant increase was shown in platelet NO and ONOO(-) levels and in iNOS and N-Tyr both in SM and in RSM pregnant women compared with controls. CONCLUSION(S): The data herein reported imply that a modified NO pathway might play a key role in the physiological changes of advancing gestation but may also contribute to the pathophysiology of spontaneous miscarriage. Thus, any factors balancing NO metabolism might be useful in the treatment of miscarriage, thus reducing the substantial morbidity and associated mortality.