Longitudinal changes in glucose metabolism in women with gestational diabetes, from late pregnancy to the postpartum period.

AIMS/HYPOTHESIS: This study aimed to determine, in women with gestational diabetes (GDM), the changes in insulin sensitivity (Matsuda Insulin Sensitivity Index; ISOGTT), insulin response and disposition index (DI) from late pregnancy (34-37 weeks gestation, T1), to early postpartum (1-5 days, T2) and late postpartum (6-12 weeks, T3). A secondary aim was to correlate the longitudinal changes in maternal lipids, adipokines, cytokines and weight in relation to the changes in ISOGTT, insulin response and DI. METHODS: ISOGTT, insulin response and DI were calculated at the three time points (T1, T2 and T3) using the results of a 75 g OGTT. Adipokines, cytokines and lipids were measured prior to each OGTT. Linear mixed-effects models were used to compare changes across each time point. Changes in ISOGTT, insulin response and DI were correlated with changes in maternal adipokines, cytokines and lipids at each time point. RESULTS: A total of 27 women completed all assessments. Compared with T1, ISOGTT was 11.20 (95% CI 8.09, 14.31) units higher at 1-5 days postpartum (p < 0.001) and was 5.49 (95% CI 2.38, 8.60) units higher at 6-12 weeks postpartum (p < 0.001). Compared with T1, insulin response values were 699.6 (95% CI 957.5, 441.6) units lower at T2 (p < 0.001) and were 356.3 (95% CI 614.3, 98.3) units lower at T3 (p = 0.004). Compared with T1, the DI was 6434.1 (95% CI 2486.2, 10,381.0) units higher at T2 (p = 0.001) and was 4262.0 (95% CI 314.6, 8209.3) units higher at T3 (p = 0.03). There was a decrease in mean cholesterol, triacylglycerol, LDL-cholesterol and VLDL-cholesterol from T1 to T2 (all p < 0.001), and an increase in mean C-reactive protein, IL-6 and IL-8 from T1 to T2 (all p < 0.001). Mean leptin decreased from T1 to T2 (p = 0.001). There was no significant change in mean adiponectin (p = 0.99) or TNF-α (p = 0.81) from T1 to T2. The mean maternal BMI decreased from T1 to T2 (p = 0.001) and T3 (p < 0.001). There were no significant correlations between any measure of change in ISOGTT, insulin response and DI and change in maternal cytokines, adipokines, lipids or weight from T1 to T2. CONCLUSIONS/INTERPRETATION: In women with GDM, delivery was associated with improvement in both insulin sensitivity and insulin production within the first few days. Improvement in insulin production persisted for 6-12 weeks, but insulin sensitivity deteriorated slightly. These changes in glucose metabolism were not associated to changes in lipids, leptin, inflammation markers or body weight. TRIAL REGISTRATION: ClinicalTrials.gov NCT02082301.

Fatty acid transporter expression and regulation is impaired in placental macrovascular endothelial cells in obese women.

OBJECTIVE: Fetal fatty acid (FA) delivery is ultimately controlled by placental transport. Focus has been the maternal-placental interface, but regulation at the feto-placental interface is unknown. METHODS: Placental macrovascular endothelial cells (EC) (n = 4/group) and trophoblasts (TB) (n = 5/group) were isolated from lean (pregravid BMI <25 kg/m2) and obese (body mass index (BMI) > 30) women. Fatty acid transporters FAT/CD36, FABPpm, FATP4, FABP 3, 4 and 5, PLIN2 and PPARα, δ, γ expression, was measured in EC and TB. Transporter response to 24 h palmitate (PA) was assessed. RESULTS: mRNA expression of FABP3, 4, 5 and PPARγ was 2- to 3-fold reduced in EC of obese versus lean women (p < .03), but not in TB. Protein level of FABPpm was 20% lower in obese (p < .05). Palmitate (PA) up-regulated CD36, FABP3, FABP4, and PLIN2 gene expression by 3- to 4-fold in lean but not obese EC (p < .05), while PA increased FABP4 and PLIN2 in lean and obese TB, and FABP5 in lean (p < .05) EC. PA exposure up-regulated peroxisome proliferator activated receptors (PPARs) 2-fold in lean and obese EC (p < .05), but not in TB. CONCLUSIONS: In obese women, FA transporter expression is lower in placental EC, but not TB, and less sensitive to saturated FA, compared to lean women. FA transport may be regulated at the feto-placental interface.

Activation of AMPK in Human Placental Explants Impairs Mitochondrial Function and Cellular Metabolism.

OBJECTIVE: Adenosine monophosphate-activated protein kinase (AMPK) is a cellular energy sensor whose phosphorylation increases energy production. We sought to evaluate the placenta-specific effect of AMPK activation on the handling of nutrients required for fetal development. METHODS: Explants were isolated from term placenta of 29 women (pregravid body mass index: 29.1 ± 9.9 kg/m2) and incubated for 24 hours with 0 to 100 µmol/L resveratrol or 0 to 1 mmol/L of 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR). Following treatment, uptake and metabolism of radiolabeled fatty acids and glucose were measured. Phosphorylation of AMPK was measured by Western blotting. Adenosine diphosphate (ATP) production was assessed using the mitochondrial ToxGlo assay kit. P < .05 was considered statistically significant. RESULTS: Resveratrol and AICAR increased AMPK phosphorylation in human placental explants. Exposure to resveratrol decreased the uptake of polyunsaturated fatty acids, arachidonic acid, and docosahexaenoic acid at 100 µmol/L ( P < .0001). Fatty acid oxidation was decreased by 100 µmol/L ( P < .05) resveratrol, while esterification was unchanged. Resveratrol decreased glucose uptake at the 50 and 100 µmol/L doses ( P < .05). Glycolysis was not significantly affected. AICAR had similar effects, decreasing fatty acid uptake and glycolysis ( P < .05). Production of ATP declined at doses found to decrease nutrient metabolism ( P < .05). CONCLUSIONS: Activation of AMPK in the human placenta leads to global downregulation of metabolism, with mitotoxicity induced at the doses of resveratrol and AICAR used to activate AMPK. Although activation of this pathway has positive metabolic effects on other tissues, in the placenta there is potential for harm, as inadequate placental delivery of critical nutrients may compromise fetal development.

Oxidative Stress Impairs Fatty Acid Oxidation and Mitochondrial Function in the Term Placenta.

Placental fatty acid oxidation (FAO) is impaired and lipid storage is increased in pregnancy states associated with chronic oxidative stress. The effect of acute oxidative stress, as seen in pregnancies complicated with asthma, on placental lipid metabolism is unknown. We hypothesized that induction of acute oxidative stress would decrease FAO and increase esterification. We assessed [3H]-palmitate oxidation and esterification in term placental explants from lean women after exposure to hydrogen peroxide (H2O2) for 4 hours. Fatty acid oxidation decreased 16% and 24% in placental explants exposed to 200 (P = .02) and 400 µM H2O2 (P = .01), respectively. Esterification was not altered with H2O2 exposure. Neither messenger RNA nor protein expression of key genes involved in FAO (eg, peroxisome proliferator-activated receptor α, carnitine palmitoyl transferase 1b) were altered. Adenosine triphosphate (ATP) levels decreased with induction of oxidative stress, without increasing cytotoxicity. Acute oxidative stress decreased FAO and ATP production in the term placenta without altering fatty acid esterification. As decreases in placental FAO and ATP production are associated with impaired fetal growth, pregnancies exposed to acute oxidative stress may be at risk for fetal growth restriction.

Effect of Maternal Obesity on Placental Lipid Metabolism.

Obese women, on average, give birth to babies with high fat mass. Placental lipid metabolism alters fetal lipid delivery, potentially moderating neonatal adiposity, yet how it is affected by maternal obesity is poorly understood. We hypothesized that fatty acid (FA) accumulation (esterification) is higher and FA ß-oxidation (FAO) is lower in placentas from obese, compared with lean women. We assessed acylcarnitine profiles (lipid oxidation intermediates) in mother-baby-placenta triads, in addition to lipid content, and messenger RNA (mRNA)/protein expression of key regulators of FA metabolism pathways in placentas of lean and obese women with normal glucose tolerance recruited at scheduled term Cesarean delivery. In isolated trophoblasts, we measured [3H]-palmitate metabolism. Placentas of obese women had 17.5% (95% confidence interval: 6.1, 28.7%) more lipid than placentas of lean women, and higher mRNA and protein expression of FA esterification regulators (e.g., peroxisome proliferator-activated receptor γ, acetyl-CoA carboxylase, steroyl-CoA desaturase 1, and diacylglycerol O-acyltransferase-1). [3H]-palmitate esterification rates were increased in trophoblasts from obese compared with lean women. Placentas of obese women had fewer mitochondria and a lower concentration of acylcarnitines, suggesting a decrease in mitochondrial FAO capacity. Conversely, peroxisomal FAO was greater in placentas of obese women. Altogether, these changes in placental lipid metabolism may serve to limit the amount of maternal lipid transferred to the fetus, restraining excess fetal adiposity in this population of glucose-tolerant women.

Effect of ω-3 supplementation on placental lipid metabolism in overweight and obese women.

BACKGROUND: The placentas of obese women accumulate lipids that may alter fetal lipid exposure. The long-chain omega-3 fatty acids (n­3 FAs) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) alter FA metabolism in hepatocytes, although their effect on the placenta is poorly understood. OBJECTIVE: We aimed to investigate whether n­3 supplementation during pregnancy affects lipid metabolism in the placentas of overweight and obese women at term. DESIGN: A secondary analysis of a double-blind randomized controlled trial was conducted in healthy overweight and obese pregnant women who were randomly assigned to DHA plus EPA (2 g/d) or placebo twice a day from early pregnancy to term. Placental FA uptake, esterification, and oxidation pathways were studied by measuring the expression of key genes in the placental tissue of women supplemented with placebo and n­3 and in vitro in isolated trophoblast cells in response to DHA and EPA treatment. RESULTS: Total lipid content was significantly lower in the placentas of overweight and obese women supplemented with n­3 FAs than in those supplemented with placebo (14.14 ± 1.03 compared with 19.63 ± 1.45 mg lipid/g tissue; P < 0.05). The messenger RNA expression of placental FA synthase (FAS) and diacylglycerol O-acyltransferase 1 (DGAT1) was negatively correlated with maternal plasma enrichment in DHA and EPA (P < 0.05). The expression of placental peroxisome proliferator­activated receptor γ (r = −0.39, P = 0.04) and its target genes DGAT1 (r = −0.37, P = 0.02) and PLIN2 (r = −0.38, P = 0.04) significantly decreased, with an increasing maternal n­3:n­6 ratio (representing the n­3 status) near the end of pregnancy. The expression of genes that regulate FA oxidation or uptake was not changed. Birth weight and length were significantly higher in the offspring of n­3-supplemented women than in those in the placebo group (P < 0.05), but no differences in the ponderal index were observed. Supplementation of n­3 significantly decreased FA esterification in isolated trophoblasts without affecting FA oxidation. CONCLUSION: Supplementing overweight and obese women with n­3 FAs during pregnancy inhibited the ability of the placenta to esterify and store lipids. This trial was registered at clinicaltrials.gov as NCT00957476.

Dietary Omega-3 Fatty Acid Supplementation Reduces Inflammation in Obese Pregnant Women: A Randomized Double-Blind Controlled Clinical Trial.

OBJECTIVE: Long-chain omega 3 fatty acids, eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) exert potent anti-inflammatory properties in humans. This study characterized the effects of omega-3 ω-3 fatty acids supplements (ω-3 FA) on the inflammatory status in the placenta and adipose tissue of overweight/obese pregnant women. STUDY DESIGN: A randomized, double-masked controlled trial was conducted in overweight/obese pregnant women that were randomly assigned to receive DHA plus EPA (2 g/day) or the equivalent of a placebo twice a day from week 10-16 to term. Inflammatory pathways were characterized in: 1) adipose tissue and placenta of treated vs. untreated women; and 2) adipose and trophoblast cells cultured with long chain FAs. RESULTS: The sum of plasma DHA and EPA increased by 5.8 fold and ω-3 FA/ω-6 FA ratio was 1.5 in treated vs. untreated women (p< 0.005). Plasma CRP concentrations were reduced (p<0.001). The adipose tissue and placenta of treated women exhibited a significant decrease in TLR4 adipose and placental expression as well as IL6, IL8, and TNFα In vitro, EPA and DHA suppressed the activation of TLR4, IL6, IL8 induced by palmitate in culture of adipose and trophoblast cells. CONCLUSION: Supplementation of overweight/obese pregnant women with dietary ω-3 FAs for >25 weeks reduced inflammation in maternal adipose and the placental tissue. TLR4 appears as a central target of the anti-inflammatory effects at the cellular level. TRIAL REGISTRATION: ClinicalTrials.gov NCT00957476.

Saturated fatty acids enhance TLR4 immune pathways in human trophoblasts.

STUDY QUESTION: What are the effects of fatty acids on placental inflammatory cytokine with respect to toll-like receptor-4/nuclear factor-kappa B (TLR4/NF-kB)? SUMMARY ANSWER: Exogenous fatty acids induce a pro-inflammatory cytokine response in human placental cells in vitro via activation of TLR4 signaling pathways. WHAT IS KNOWN ALREADY: The placenta is exposed to changes in circulating maternal fatty acid concentrations throughout pregnancy. Fatty acids are master regulators of innate immune pathways through recruitment of toll-like receptors and activation of cytokine synthesis. STUDY DESIGN, SIZE, DURATION: Trophoblast cells isolated from 14 normal term human placentas were incubated with long chain fatty acids (FA) of different carbon length and degree of saturation. The expression and secretion of interleukin-6 (IL-6), IL-8 and tumor necrosis factor-alpha (TNF-α) were measured by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Antibodies against TLR4 ligand binding domain, downstream signaling and anti-p65 NFkB-inhibitor were used to characterize the pathways of FA action. PARTICIPANTS/MATERIALS, SETTING, METHODS: General approach used primary human term trophoblast cell culture. Methods and end-points used real-time quantitative PCR, cytokine measurements, immunohistochemistry, western blots. MAIN RESULTS AND THE ROLE OF CHANCE: The long chain saturated fatty acids, stearic and palmitic (PA), stimulated the synthesis as well as the release of TNF-α, IL-6 and IL-8 by trophoblast cells (2- to 6-fold, P < 0.001). In contrast, the unsaturated (palmitoleic, oleic, linoleic) acids did not modify cytokine expression significantly. Palmitate-induced inflammatory effects were mediated via TLR4 activation, NF-kB phosphorylation and nuclear translocation. LIMITATIONS, REASONS FOR CAUTION: TNF-α protein level was close to the limit of detection in the culture medium even when cells were cultured with PA. WIDER IMPLICATIONS OF THE FINDINGS: These mechanisms open the way to a better understanding of how changes in maternal lipid homeostasis may regulate placental inflammatory status. STUDY FUNDING/COMPETING INTERESTS: X.Y. was recipient of fellowship award from West China Second University Hospital, Sichuan University (NIH HD 22965-19). The authors have nothing else to disclose. TRIAL REGISTRATION NUMBER: None.

Identification of early transcriptome signatures in placenta exposed to insulin and obesity.

OBJECTIVE: The purpose of this study was to investigate the effects of insulin on human placental transcriptome and biological processes in first-trimester pregnancy. STUDY DESIGN: Maternal plasma and placenta villous tissue were obtained at the time of voluntary termination of pregnancy (7-12 weeks) from 17 lean (body mass index, 20.9±1.5 kg/m2) and 18 obese (body mass index, 33.5±2.6 kg/m2) women. Trophoblast cells were immediately isolated for in vitro treatment with insulin or vehicle. Patterns of global gene expression were analyzed using genome microarray profiling after hybridization to Human Gene 1.1 ST and real time reverse transcription-polymerase chain reaction. RESULTS: The global trophoblast transcriptome was qualitatively separated in insulin-treated vs untreated trophoblasts of lean women. The number of insulin-sensitive genes detected in the trophoblasts of lean women was 2875 (P<.001). Maternal obesity reduced the number of insulin-sensitive genes recovered by 30-fold. Insulin significantly impaired several gene networks regulating cell cycle and cholesterol homeostasis but did not modify pathways related to glucose transport. Obesity associated with high insulin and insulin resistance, but not maternal hyperinsulinemia alone, impaired the global gene profiling of early gestation placenta, highlighting mitochondrial dysfunction and decreased energy metabolism. CONCLUSION: We report for the first time that human trophoblast cells are highly sensitive to insulin regulation in early gestation. Maternal obesity associated with insulin resistance programs the placental transcriptome toward refractoriness to insulin with potential adverse consequences for placental structure and function.

Obesity-induced down-regulation of the mitochondrial translocator protein (TSPO) impairs placental steroid production.

CONTEXT: Low concentrations of estradiol and progesterone are hallmarks of adverse pregnancy outcomes as is maternal obesity. During pregnancy, placental cholesterol is the sole source of sex steroids. Cholesterol trafficking is the limiting step in sex steroid biosynthesis and is mainly mediated by the translocator protein (TSPO), present in the mitochondrial outer membrane. OBJECTIVE: The objective of the study was to investigate the effects of maternal obesity in placental sex steroid biosynthesis and TSPO regulation. DESIGN/PARTICIPANTS: One hundred forty-four obese (body mass index 30-35 kg/m(2)) and 90 lean (body mass index 19-25 kg/m(2)) pregnant women (OP and LP, respectively) recruited at scheduled term cesarean delivery. Placenta and maternal blood were collected. SETTING: This study was conducted at MetroHealth Medical Center (Cleveland, Ohio). MAIN OUTCOME MEASURES: Maternal metabolic components (fasting glucose, insulin, leptin, estradiol, progesterone, and total cholesterol) and placental weight were measured. Placenta (mitochondria and membranes separated) and cord blood cholesterol values were verified. The expression and regulation of TSPO and mitochondrial function were analyzed. RESULTS: Plasma estradiol and progesterone concentrations were significantly lower (P < .04) in OP as compared with LP women. Maternal and cord plasma cholesterol were not different between groups. Placental citrate synthase activity and mitochondrial DNA, markers of mitochondrial density, were unchanged, but the mitochondrial cholesterol concentrations were 40% lower in the placenta of OP. TSPO gene and protein expressions were decreased 2-fold in the placenta of OP. In vitro trophoblast activation of the innate immune pathways with lipopolysaccharide and long-chain saturated fatty acids reduced TSPO expression by 2- to 3-fold (P < .05). CONCLUSION: These data indicate that obesity in pregnancy impairs mitochondrial steroidogenic function through the negative regulation of mitochondrial TSPO.

The altered mononuclear cell-derived cytokine response to glucose ingestion is not regulated by excess adiposity in polycystic ovary syndrome.

CONTEXT: Excess adipose tissue is a source of inflammation. Polycystic ovary syndrome (PCOS) is a proinflammatory state and is often associated with excess abdominal adiposity (AA) alone and/or frank obesity. OBJECTIVE: To determine the effect of glucose ingestion on cytokine release from mononuclear cells (MNC) in women with PCOS with and without excess AA and/or obesity. DESIGN: A cross-sectional study. SETTING: Academic medical center. PATIENTS: Twenty-three women with PCOS (seven normal weight with normal AA, eight normal weight with excess AA, eight obese) and 24 ovulatory controls (eight normal weight with normal AA, eight normal weight with excess AA, eight obese). INTERVENTION: Three-hour 75-g oral glucose tolerance test (OGTT). MAIN OUTCOME MEASURES: Body composition was measured by dual energy x-ray absorptiometry. Insulin sensitivity was derived from the OGTT (ISOGTT). TNFα, IL-6, and IL-1ß release was measured in supernatants of cultured MNC isolated from blood samples drawn while fasting and 2 hours after glucose ingestion. RESULTS: Insulin sensitivity was lower in obese subjects regardless of PCOS status and in normal-weight women with PCOS compared with normal-weight controls regardless of body composition status. In response to glucose ingestion, MNC-derived TNFα, IL-6, and IL-1ß release decreased in both normal-weight control groups but failed to suppress in either normal-weight PCOS group and in obese women regardless of PCOS status. For the combined groups, the cytokine responses were negatively correlated with insulin sensitivity and positively correlated with abdominal fat and androgens. CONCLUSIONS: Women with PCOS fail to suppress MNC-derived cytokine release in response to glucose ingestion, and this response is independent of excess adiposity. Nevertheless, a similar response is also a feature of obesity per se. Circulating MNC and excess adipose tissue are separate and distinct sources of inflammation in this population.

Evidence of mononuclear cell preactivation in the fasting state in polycystic ovary syndrome.

OBJECTIVE: We evaluated mononuclear cell (MNC) preactivation in women with polycystic ovary syndrome (PCOS) by examining the effect of in vitro lipopolysaccharide (LPS) exposure on cytokine release in the fasting state. STUDY DESIGN: Twenty women with PCOS (10 lean, 10 obese) and 20 weight-matched controls (10 lean, 10 obese) volunteered for study participation. Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) release was measured from mononuclear cells isolated from fasting blood samples and cultured in the presence and absence of LPS. Plasma IL-6 was measured from the same fasting blood samples. Insulin sensitivity was derived from an oral glucose tolerance test using the Matsuda index, and truncal fat was measured by dual-energy x-ray absorptiometry. RESULTS: The percent change from baseline in TNF-α and IL-6 release from MNC following LPS exposure was increased (P < .04) in lean and obese women with PCOS and obese controls compared with lean controls. Plasma IL-6 was increased (P < .02) in obese women with PCOS compared with lean women with PCOS, which in turn was increased (P < .02) compared with lean controls. The MNC-derived TNF-α and IL-6 responses from MNCs were negatively correlated with insulin sensitivity (P < .03) and positively correlated with testosterone (P < .03) and androstenedione (P < .006) for the combined groups. Plasma IL-6 was positively correlated with percentage truncal fat (P < .008). CONCLUSION: In PCOS, increased cytokine release from MNCs following LPS exposure in the fasting state reveals the presence of MNC preactivation. Importantly, this phenomenon is independent of obesity and may contribute to the development of insulin resistance and hyperandrogenism in PCOS. In contrast, the source of plasma IL-6 elevations in PCOS may be excess adiposity.

Sex-specific effects of maternal anthropometrics on body composition at birth.

OBJECTIVE: The purpose of this study was to assess whether maternal factors that are associated with fetal lean and fat mass differ between sexes. STUDY DESIGN: Secondary analysis of a prospective cohort that delivered by scheduled cesarean section from 2004-2013. Maternal blood was collected before surgery for metabolic parameters. Placental weight and neonatal anthropometrics were measured within 48 hours. Anthropometric differences between sexes were assessed with the Student t test. Multiple stepwise regression analysis assessed the relationship between independent maternal variables and neonatal lean body mass (LBM), fat mass (FM), or percentage of fat as dependent variables in male and female infants combined and separately. RESULTS: We analyzed 360 women with normal glucose tolerance and a wide range of pregravid body mass index (16-64 kg/m(2)) and their offspring (male, 194; female, 166). Male infants had more FM (mean difference, 40 ± 18 g; P = .03) and LBM (mean difference, 158 ± 34 g; P < .0001) than female infants. Percentage of body fat and measured maternal variables did not differ between sexes. In both sexes, placental weight had the strongest correlation with both neonatal LBM and FM, which accounted for 20-39% of the variance. In male infants, maternal height, body mass index, and weight gain were significant predictors of both lean and fat mass. In female infants, plasma interleukin-6 and C-reactive protein, respectively, were associated independently with percentage of body fat and LBM. CONCLUSION: Our findings suggest that the body composition and inflammatory environment of the mother modulate the metabolic fitness of neonates, as predicted by fat and lean mass, in a sex-specific manner.

Glucose and lipopolysaccharide regulate proatherogenic cytokine release from mononuclear cells in polycystic ovary syndrome.

Women with polycystic ovary syndrome (PCOS) have chronic low-grade inflammation, which can increase the risk of atherogenesis. We examined the effect of glucose ingestion and lipopolysaccharide (LPS) on markers of proatherogenic inflammation in the mononuclear cells (MNC) and plasma of women with PCOS. Sixteen women with PCOS (8 lean, 8 obese) and 15 weight-matched controls (8 lean, 7 obese) underwent a 3-h oral glucose tolerance test (OGTT). Interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) release from MNC cultured in the presence of LPS and plasma IL-6, C-reactive protein (CRP), and soluble vascular adhesion molecule-1 (sVCAM-1) were measured from blood samples drawn while fasting and 2h after glucose ingestion. Truncal fat was measured by dual-energy absorptiometry (DEXA). Lean women with PCOS and obese controls failed to suppress LPS-stimulated IL-6 and IL-1ß release from MNC after glucose ingestion. In contrast, obese women with PCOS suppressed these MNC-derived cytokines under the same conditions. In response to glucose ingestion, plasma IL-6 and sVCAM-1 increased and CRP suppression was attenuated in both PCOS groups and obese controls compared with lean controls. Fasting plasma IL-6 and CRP correlated positively with percentage of truncal fat. The absolute change in plasma IL-6 correlated positively with testosterone. We conclude that glucose ingestion promotes proatherogenic inflammation in PCOS with a systemic response that is independent of obesity. Based on the suppressed MNC-derived cytokine responses suggestive of LPS tolerance, chronic low-grade inflammation may be more profound in obese women with PCOS. Excess abdominal adiposity and hyperandrogenism may contribute to atherogenesis in PCOS.

Elevated circulating levels of tissue factor in polycystic ovary syndrome.

Women with polycystic ovary syndrome (PCOS) have coagulation disturbances and inflammation, which increases the risk of atherothrombosis. We evaluated the status of circulating tissue factor (TF), the receptor for coagulation factor VII involved in atherothrombosis, in women with PCOS and weight-matched controls. Two-way analysis of variance models were fit to evaluate the effect of PCOS status and weight class on TF and other parameters. The TF levels were significantly higher in lean women with PCOS compared to lean controls. Plasminogen activator inhibitor 1 (PAI-1) levels were significantly higher in obese participants compared to lean participants after controlling for PCOS status. The TF levels directly correlated with percentage of truncal fat and plasma levels of PAI-1, testosterone, androstendione, and dehydroepiandrosterone-sulfate; and inversely correlated with insulin sensitivity index-OGTT(IS(OGTT)). Circulating TF is elevated in PCOS independent of obesity, but both PCOS and obesity contribute to a prothrombotic state. In PCOS, abdominal adiposity and hyperandrogenism may exacerbate the risk of atherothrombosis.

Glucose ingestion stimulates atherothrombotic inflammation in polycystic ovary syndrome.

Women with polycystic ovary syndrome (PCOS) have chronic low-grade inflammation that can increase the risk of atherothrombosis. We performed a cross-sectional study to examine the effect of glucose ingestion on markers of atherothrombotic inflammation in mononuclear cells (MNC) of 16 women with PCOS (8 lean, 8 obese) and 16 weight-matched controls. Activator protein-1 (AP-1) activation and the protein content of early growth response-1 (EGR-1), matrix matalloproteinases-2 (MMP2), and tissue factor (TF) were quantified from MNC obtained from blood drawn fasting and 2 h after glucose ingestion. Plasma MMP9 and C-reactive protein (CRP) were measured from fasting blood samples. Truncal fat was determined by DEXA. Lean women with PCOS exhibited greater AP-1 activation and MMP2 protein content after glucose ingestion and higher plasma MMP9 and CRP levels than lean controls. Obese women with PCOS exhibited greater EGR-1 and TF protein content after glucose ingestion, and plasma CRP levels were even higher compared with lean subjects regardless of PCOS status. Truncal fat correlated with MMP9 and CRP levels and glucose-stimulated increases in AP-1 activation and EGR-1 and TF protein content. Testosterone correlated with glucose-stimulated AP-1 activation, and androstenedione correlated with MMP9 and CRP levels and glucose-stimulated AP-1 activation. Thus, both PCOS and obesity contribute to an atherothrombotic state in which excess abdominal adiposity and hyperandrogenism may be specific risk factors for developing atherothrombosis.

Hyperglycemia-induced oxidative stress is independent of excess abdominal adiposity in normal-weight women with polycystic ovary syndrome.

STUDY QUESTION: What is the effect of glucose ingestion on leukocytic reactive oxygen species (ROS) generation in normal-weight women with polycystic ovary syndrome (PCOS) with and without excess abdominal adiposity (AA)? SUMMARY ANSWER: Normal-weight women with PCOS exhibit an increase in leukocytic ROS generation in response to glucose ingestion, and this increase is independent of excess AA. WHAT IS KNOWN ALREADY: Excess adipose tissue is a source of oxidative stress. Normal-weight women with PCOS exhibit oxidative stress and can have excess AA. STUDY DESIGN AND SIZE: This is a cross-sectional study involving 30 reproductive-age women. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Fourteen normal-weight women with PCOS (6 normal AA, 8 excess AA) and 16 body composition-matched controls (8 normal AA, 8 excess AA) underwent body composition assessment by dual-energy absorptiometry and an oral glucose tolerance test (OGTT) at a university medical center. Insulin sensitivity was derived from the OGTT (IS(OGTT)). Blood was drawn while fasting and 2 h after glucose ingestion to measure leukocytic ROS generation and p47(phox) protein content and plasma thiobarbituric acid-reactive substances (TBARS) and C-reactive protein (CRP). MAIN RESULTS AND THE ROLE OF CHANCE: Compared with controls, both PCOS groups exhibited lower IS(OGTT) (43-54%) and greater percentage change (% change) in ROS generation (96-140%), p47(phox) protein (18-28%) and TBARS (17-48%). Compared with women with PCOS with excess AA, those with normal AA exhibited higher testosterone levels (29%) and lower CRP levels (70%). For the combined groups, IS(OGTT) was negatively correlated with the % change in ROS generation and p47(phox) protein. CRP was positively correlated with abdominal fat. The % change in p47(phox) protein was positively correlated with CRP and androgens. LIMITATIONS, REASONS FOR CAUTION: Although this study is adequately powered to assess differences in ROS generation between the women with PCOS and control participants, the modest sample size merits caution when interpreting the corroborative results of the additional measures of oxidative stress and inflammation. WIDER IMPLICATIONS OF THE FINDINGS: This study highlights the unique pro-oxidant contribution of circulating leukocytes in the development of insulin resistance and hyperandrogenism in PCOS. STUDY FUNDING/COMPETING INTEREST(S): Supported by NIH grant HD-048535 to F.G. The authors have nothing to disclose.

Inflammation in response to glucose ingestion is independent of excess abdominal adiposity in normal-weight women with polycystic ovary syndrome.

CONTEXT: Inflammation and excess abdominal adiposity (AA) are often present in normal-weight women with polycystic ovary syndrome (PCOS). OBJECTIVE: We determined the effects of hyperglycemia on nuclear factor-κB (NFκB) activation in mononuclear cells (MNC) of normal-weight women with PCOS with and without excess AA. DESIGN: This was a prospective controlled study. SETTING: The study was conducted at an academic medical center. PATIENTS: Fifteen normal-weight, reproductive-age women with PCOS (seven normal AA, eight excess AA) and 16 body composition-matched controls (eight normal AA, eight excess AA) participated in the study. MAIN OUTCOME MEASURES: Body composition was measured by dual-energy absorptiometry. Insulin sensitivity was derived from an oral glucose tolerance test (IS(OGTT)). Activated NFκB and the protein content of p65 and inhibitory-κB were quantified from MNC, and TNFα and C-reactive protein (CRP) were measured in plasma obtained from blood drawn while fasting and 2 h after glucose ingestion. RESULTS: Compared with controls, both PCOS groups exhibited lower IS(OGTT), increases in activated NFκB and p65 protein, and decreases in inhibitory-κB protein. Compared with women with PCOS with excess AA, those with normal AA exhibited higher testosterone levels and lower TNFα and CRP levels. For the combined groups, the percent change in NFκB activation was negatively correlated with IS(OGTT) and positively correlated with androgens. TNFα and CRP were positively correlated with abdominal fat. CONCLUSION: In normal-weight women with PCOS, the inflammatory response to glucose ingestion is independent of excess AA. Circulating MNC and excess AA are separate and unique sources of inflammation in this population.

Molecular inflammation and adipose tissue matrix remodeling precede physiological adaptations to pregnancy.

Changes in adipose tissue metabolism are central to adaptation of whole body energy homeostasis to pregnancy. To gain insight into the molecular mechanisms supporting tissue remodeling, we have characterized the longitudinal changes of the adipose transcriptome in human pregnancy. Healthy nonobese women recruited pregravid were followed in early (8-12 wk) and in late (36-38 wk) pregnancy. Adipose tissue biopsies were obtained in the fasting state from the gluteal depot. The adipose transcriptome was examined via whole genome DNA microarray. Expression of immune-related genes and extracellular matrix components was measured using real-time RT-PCR. Adipose mass, adipocyte size, and cell number increased in late pregnancy compared with pregravid measurements (P < 0.001) but remained unchanged in early pregnancy. The adipose transcriptome evolved during pregnancy with 10-15% of genes being differently expressed compared with pregravid. Functional gene cluster analysis revealed that the early molecular changes affected immune responses, angiogenesis, matrix remodeling, and lipid biosynthesis. Increased expression of macrophage markers (CD68, CD14, and the mannose-6 phosphate receptor) emphasized the recruitment of the immune network in both early and late pregnancy. The TLR4/NF-κB signaling pathway was enhanced specifically in relation to inflammatory adipokines and chemokines genes. We conclude that early recruitment of metabolic and immune molecular networks precedes the appearance of pregnancy-related physiological changes in adipose tissue. This biphasic pattern suggests that physiological inflammation is an early step preceding the development of insulin resistance, which peaks in late pregnancy.

Molecular phenotype of monocytes at the maternal-fetal interface.

OBJECTIVE: The purpose of this study was to gain insight into the pathways that are associated with inflammation at the maternal-fetal interface. This study examined the molecular characteristics of monocytes that were derived from the maternal circulation and the placenta of obese women. STUDY DESIGN: Mononuclear cells were isolated from placenta, venous maternal, and umbilical cord blood at term delivery; activated monocytes were separated with CD14 immunoselection. The genotype and expression pattern of the monocytes were analyzed by microarray and real-time reverse transcriptase-polymerase chain reaction. RESULTS: The transcriptome of the maternal blood and placental CD14 monocytes exhibited 73% homology, with 10% (1800 common genes) differentially expressed. Genes for immune sensing and regulation, matrix remodeling, and lipid metabolism were enhanced 2-2006 fold in placenta, compared with maternal monocytes. The CD14 placental monocytes exhibited a maternal genotype (9% DYS14 expression) as opposed to the fetal genotype (90% DYS14 expression) of the trophoblast cells. CONCLUSION: CD14 monocytes from the maternal blood and the placenta share strong phenotypic and genotypic similarities with an enhanced inflammatory pattern in the placenta. The functional traits of the CD14 blood and placental monocytes suggest that they both contribute to propagation of inflammation at the maternal-fetal interface.