Bogdan Panaitescu

Metformin, the aspirin of the 21st century: its role in gestational diabetes mellitus, prevention of preeclampsia and cancer, and the promotion of longevity

&NA; Metformin is everywhere. Originally introduced in clinical practice as an antidiabetic agent, its role as a therapeutic agent is expanding to include treatment of prediabetes mellitus, gestational diabetes mellitus, and polycystic ovarian disease; more recently, experimental studies and observations in randomized clinical trials suggest that metformin could have a place in the treatment or prevention of preeclampsia. This article provides a brief overview of the history of metformin in the treatment of diabetes mellitus and reviews the results of metaanalyses of metformin in gestational diabetes mellitus as well as the treatment of obese, non‐diabetic, pregnant women to prevent macrosomia. We highlight the results of a randomized clinical trial in which metformin administration in early pregnancy did not reduce the frequency of large‐for‐gestational‐age infants (the primary endpoint) but did decrease the frequency of preeclampsia (a secondary endpoint). The mechanisms by which metformin may prevent preeclampsia include a reduction in the production of antiangiogenic factors (soluble vascular endothelial growth factor receptor‐1 and soluble endoglin) and the improvement of endothelial dysfunction, probably through an effect on the mitochondria. Another potential mechanism whereby metformin may play a role in the prevention of preeclampsia is its ability to modify cellular homeostasis and energy disposition, mediated by rapamycin, a mechanistic target. Metformin has a molecular weight of 129 Daltons and therefore readily crosses the placenta. There is considerable evidence to suggest that this agent is safe during pregnancy. New literature on the role of metformin as a chemotherapeutic adjuvant in the prevention of cancer and in prolonging life and protecting against aging is reviewed briefly. Herein, we discuss the mechanisms of action and potential benefits of metformin.

Intra-Amniotic Administration of HMGB1 Induces Spontaneous Preterm Labor and Birth

Sterile intra‐amniotic inflammation is associated with spontaneous preterm labor. Alarmins are proposed to mediate this inflammatory process. The aim of this study was to determine whether intra‐amniotic administration of an alarmin, HMGB1, could induce preterm labor/birth.

Invariant NKT cell activation induces late preterm birth that is attenuated by rosiglitazone

Preterm birth (PTB) is the leading cause of neonatal morbidity and mortality worldwide. Although intra-amniotic infection is a recognized cause of spontaneous preterm labor, the noninfection-related etiologies are poorly understood. In this article, we demonstrated that the expansion of activated CD1d-restricted invariant NKT (iNKT) cells in the third trimester by administration of α-galactosylceramide (α-GalCer) induced late PTB and neonatal mortality. In vivo imaging revealed that fetuses from mice that underwent α-GalCer–induced late PTB had bradycardia and died shortly after delivery. Yet, administration of α-GalCer in the second trimester did not cause pregnancy loss. Peroxisome proliferator–activated receptor (PPAR)γ activation, through rosiglitazone treatment, reduced the rate of α-GalCer–induced late PTB and improved neonatal survival. Administration of α-GalCer in the third trimester suppressed PPARγ activation, as shown by the downregulation of Fabp4 and Fatp4 in myometrial and decidual tissues, respectively; this suppression was rescued by rosiglitazone treatment. Administration of α-GalCer in the third trimester induced an increase in the activation of conventional CD4+ T cells in myometrial tissues and the infiltration of activated macrophages, neutrophils, and mature dendritic cells to myometrial and/or decidual tissues. All of these effects were blunted after rosiglitazone treatment. Administration of α-GalCer also upregulated the expression of inflammatory genes at the maternal–fetal interface and systemically, and rosiglitazone treatment partially attenuated these responses. Finally, an increased infiltration of activated iNKT-like cells in human decidual tissues is associated with noninfection-related preterm labor/birth. Collectively, these results demonstrate that iNKT cell activation in vivo leads to late PTB by initiating innate and adaptive immune responses and suggest that the PPARγ pathway has potential as a target for prevention of this syndrome.

A Role for the Inflammasome in Spontaneous Preterm Labor With Acute Histologic Chorioamnionitis

Inflammasomes are cytosolic multiprotein complexes that orchestrate inflammation in response to pathogens and endogenous danger signals. Herein, we determined whether the chorioamniotic membranes from women in spontaneous preterm labor with acute histologic chorioamnionitis (1) express major inflammasome components; (2) express caspase (CASP)-1 and CASP-4 as well as their active forms; (3) exhibit apoptosis-associated speck-like protein containing a CARD (ASC)/CASP-1 complex formation; and (4) release the mature forms of interleukin (IL)-1β and IL-18. We utilized quantitative reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, immunoblotting, and immunohistochemistry to determine the messenger RNA (mRNA) and protein expression of major inflammasome components, nucleotide-binding oligomerization domain (NOD) proteins, and the pro- and mature/active forms of CASP-1, CASP-4, IL-1β, and IL-18. The ASC/CASP-1 complex formation was determined using an in situ proximity ligation assay. When comparing the chorioamniotic membranes from women in spontaneous preterm labor with acute histologic chorioamnionitis to those without this placental lesion, we found that (1) the mRNA of NLR family pyrin domain-containing protein (NLRP)1, NLRP3, NLR family CARD domain-containing protein 4 (NLRC4), and NOD2 were higher; (2) the NLRP3 protein was increased; (3) the mRNA and active form (p10) of CASP-1 were greater; (4) the mRNA and active form of CASP-4 were increased; (5) the mRNA and mature form of IL-1β were higher; (6) the mature form of IL-18 was elevated; and (7) ASC/CASP-1 complex formation was increased. In conclusion, spontaneous preterm labor with acute histologic chorioamnionitis is characterized by an upregulation of NLRP3 and the active form of CASP-4, as well as increased ASC/CASP-1 complex formation, which may participate in the activation of CASP-1 and the maturation of IL-1β and IL-18 in the chorioamniotic membranes. These findings provide the first evidence that supports a role for the inflammasome in the pathological inflammation implicated in spontaneous preterm labor with acute histologic chorioamnionitis.

Are amniotic fluid neutrophils in women with intraamniotic infection and/or inflammation of fetal or maternal origin?

Background Neutrophils are the most abundant white blood cells found in the amniotic cavity of women with intraamniotic infection and/or inflammation. The current belief is that these neutrophils are of fetal origin. However, abundant neutrophils have been found in the amniotic fluid of women with a severe acute maternal inflammatory response but without a severe fetal inflammatory response in the placenta, suggesting that these innate immune cells can also be of maternal origin or a mixture of both fetal and maternal neutrophils. Objective We sought to investigate the origin of amniotic fluid neutrophils from women with intraamniotic infection and/or inflammation and to correlate these findings with acute histologic maternal and fetal inflammatory responses in the placenta. Study Design Amniotic fluid was collected from 15 women with suspected intraamniotic infection and/or inflammation (positive microbiological cultures and/or interleukin‐6 concentrations ≥2.6 ng/mL). Amniotic fluid neutrophils were purified by fluorescence‐activated cell sorting, DNA was extracted, and DNA fingerprinting was performed. DNA fingerprinting was also performed in the umbilical cord and maternal blood DNA. Fluorescence in situ hybridization was assayed in women with male neonates. Blinded placental histopathological evaluations were conducted. Results First, DNA fingerprinting revealed that 43% (6/14) of women who underwent a single amniocentesis had mostly fetal neutrophils in the amniotic fluid. Second, DNA fingerprinting showed that 36% (5/14) of the women who underwent a single amniocentesis had predominantly maternal neutrophils in the amniotic fluid. Third, DNA fingerprinting indicated that 21% (3/14) of the women who underwent a single amniocentesis had an evident mixture of fetal and maternal neutrophils in the amniotic fluid. Fourth, DNA fingerprinting revealed that a woman who underwent 2 amniocenteses (patient 15) had fetal neutrophils first, and as infection progressed, abundant maternal neutrophils invaded the amniotic cavity. Fifth, fluorescence in situ hybridization confirmed DNA fingerprinting results by showing that both fetal and maternal neutrophils were present in the amniotic fluid. Sixth, most of the women who had predominantly amniotic fluid neutrophils of fetal origin at the time of collection delivered extremely preterm neonates (71% [5/7]). Seventh, all of the women who had predominantly amniotic fluid neutrophils of maternal origin at the time of collection delivered term or late preterm neonates (100% [6/6]). Eighth, 2 of the women who had an evident mixture of fetal and maternal neutrophils in the amniotic fluid at the time of collection delivered extremely preterm neonates (67% [2/3]), and the third woman delivered a term neonate (33% [1/3]). Finally, most of the women included in this study presented acute maternal and fetal inflammatory responses in the placenta (87% [13/15]). Conclusion Amniotic fluid neutrophils can be either predominantly of fetal or maternal origin, or a mixture of both fetal and maternal origin, in women with intraamniotic infection and/or inflammation. The findings herein provide evidence that both fetal and maternal neutrophils can invade the amniotic cavity, suggesting that both the fetus and the mother participate in the host defense mechanisms against intraamniotic infection.

Twenty-four percent of patients with clinical chorioamnionitis in preterm gestations have no evidence of either culture-proven intraamniotic infection or intraamniotic inflammation

BACKGROUND: Recent studies on clinical chorioamnionitis at term suggest that some patients with this diagnosis have neither intraamniotic infection nor intraamniotic inflammation. A false‐positive diagnosis of clinical chorioamnionitis in preterm gestation may lead to unwarranted preterm delivery. OBJECTIVE: We sought to determine the frequency of intraamniotic inflammation and microbiologically proven amniotic fluid infection in patients with preterm clinical chorioamnionitis. STUDY DESIGN: Amniocentesis was performed in singleton pregnant women with preterm clinical chorioamnionitis (<36 weeks of gestation). Amniotic fluid was cultured for aerobic and anaerobic bacteria and genital mycoplasmas and assayed for matrix metalloproteinase‐8 concentration. Microbial invasion of the amniotic cavity was defined as a positive amniotic fluid culture; intraamniotic inflammation was defined as an elevated amniotic fluid matrix metalloproteinase‐8 concentration of >23 ng/mL. Nonparametric and survival techniques were used for analysis. RESULTS: Among patients with preterm clinical chorioamnionitis, 24% (12/50) had neither microbiologic evidence of intraamniotic infection nor intraamniotic inflammation. Microbial invasion of the amniotic cavity was present in 34% (18/53) and intraamniotic inflammation in 76% (38/50) of patients. The most common microorganisms isolated from the amniotic cavity were the Ureaplasma species. Finally, patients without microbial invasion of the amniotic cavity or intraamniotic inflammation had significantly lower rates of adverse outcomes (including lower gestational age at delivery, a shorter amniocentesis‐to‐delivery interval, acute histologic chorioamnionitis, acute funisitis, and significant neonatal morbidity) than those with microbial invasion of the amniotic cavity and/or intraamniotic inflammation. CONCLUSION: Among patients with preterm clinical chorioamnionitis, 24% had no evidence of either intraamniotic infection or intraamniotic inflammation, and 66% had negative amniotic fluid cultures, using standard microbiologic techniques. These observations call for a reexamination of the criteria used to diagnose preterm clinical chorioamnionitis.

Neutrophil Extracellular Traps in the Amniotic Cavity of Women with Intra-Amniotic Infection A New Mechanism of Host Defense

Objective: Neutrophil extracellular traps (NETs) control microbial infections through their antimicrobial activities attributed to DNA, histones, granules, and cytoplasmic proteins (eg, elastase). Intra-amniotic infection is characterized by the influx of neutrophils into the amniotic cavity; therefore, the aim of this study was to determine whether amniotic fluid neutrophils form NETs in this inflammatory process. Methods: Amniotic fluid samples from women with intra-amniotic infection (n = 15) were stained for bacteria detection using fluorescent dyes. Amniotic fluid neutrophils were purified by filtration. As controls, neutrophils from maternal blood samples (n = 3) were isolated by density gradients. Isolated neutrophils were plated onto glass cover slips for culture with and without 100 nM of phorbol-12-myristate-13-acetate (PMA). NET formation was assessed by 4’,6-diamidino-2-phenylindole (DAPI) staining and scanning electron microscopy. Different stages of NET formation were visualized using antibodies against elastase and histone H3, in combination with DAPI staining, by confocal microscopy. Finally, maternal or neonatal neutrophils were added to amniotic fluid samples from women without intra-amniotic infection (n = 4), and NET formation was evaluated by DAPI staining. Results: (1) NETs were present in the amniotic fluid of women with intra-amniotic infection; (2) all of the amniotic fluid samples had detectable live and dead bacteria associated with the presence of NETs; (3) in contrast to neutrophils from the maternal circulation, amniotic fluid neutrophils did not require PMA stimulation to form NETs; (4) different stages of NET formation were observed by co-localizing elastase, histone H3, and DNA in amniotic fluid neutrophils; and (5) neither maternal nor neonatal neutrophils form NETs in the amniotic fluid of women without intra-amniotic infection. Conclusion: NETs are detectable in the amniotic fluid of women with intra-amniotic infection.

Amniotic fluid neutrophils can phagocytize bacteria: A mechanism for microbial killing in the amniotic cavity

Neutrophils are capable of performing phagocytosis, a primary mechanism for microbial killing. Intra‐amniotic infection is characterized by an influx of neutrophils into the amniotic cavity. Herein, we investigated whether amniotic fluid neutrophils could phagocytize bacteria found in the amniotic cavity of women with intra‐amniotic infection.

In vivo T-cell activation by a monoclonal αCD3ε antibody induces preterm labor and birth.

Activated/effector T cells seem to play a role in the pathological inflammation associated with preterm labor. The aim of this study was to determine whether in vivo T‐cell activation by a monoclonal αCD3ε antibody induces preterm labor and birth.

Inflammasome assembly in the chorioamniotic membranes during spontaneous labor at term

Inflammasome activation requires two steps: priming and assembly of the multimeric complex. The second step includes assembly of the sensor molecule and adaptor protein ASC (an apoptosis‐associated speck‐like protein containing a CARD), which results in ASC speck formation and the recruitment of caspase (CASP)‐1. Herein, we investigated whether there is inflammasome assembly in the chorioamniotic membranes and choriodecidual leukocytes from women who underwent spontaneous labor at term.