Patricia K. Senagore

Mid-pregnancy circulating cytokine levels, histologic chorioamnionitis and spontaneous preterm birth

Some spontaneous preterm deliveries (PTD) are caused by occult infections of the fetal membranes (histologic chorioamnionitis [HCA]). High levels of infection-related markers, including some cytokines, sampled from maternal circulation in mid-pregnancy have been linked to PTD, but whether these specifically identify HCA has not been established. We have tested associations between 13 Th1, Th2 and Th17 cytokines and PTD with and without HCA in a prospective cohort study. The study sample included 926 Pregnancy Outcomes and Community Health Study subcohort women; women with medically indicated PTD or incomplete data excluded. A panel of cytokines was assessed using a multiplex assay in maternal plasma collected at 15-27 weeks of gestation. Severe HCA was scored by a placental pathologist blinded to clinical variables. Multivariable polytomous logistic regression was used to estimate adjusted odds ratios (OR) per 1 standard deviation (S.D.) increase in cytokine levels using a 5 level outcome variable: PTD <35 weeks with HCA, PTD <35 weeks without HCA, PTD 35-36 weeks with HCA, PTD 35-36 weeks without HCA, and term (referent). Interleukin (IL)-1beta, IL-2, IL-12, interferon-gamma, IL-4, IL-6 and transforming growth factor-beta were all significantly associated with PTD <35 weeks with HCA, with ORs of 1.6-2.3 per S.D. increase. None of these were associated with PTD <35 weeks without HCA or PTD 35-36 weeks with HCA. Although the tissues of origin of circulating cytokines are unclear, the observed elevations across many cytokines among women who later delivered <35 weeks with HCA may represent a robust immune response to infection within gestational tissues. These results suggest that women with HCA could be identified using relatively non-invasive means.

Histological characteristics of singleton placentas delivered before the 28th week of gestation

Aims: The placenta is a record of the fetal environment and its examination may provide information about the babys subsequent growth and development. We describe the histological characteristics of 947 singleton placentas from infants born between 23 and 27 weeks gestation. Methods: Consent was obtained from mothers who delivered before 28 weeks (clinical estimate). We evaluated the gross and histopathological features of the placenta and assessed pair‐wise correlations between variables. Results: Lesions of uteroplacental circulation (abruption, extensive infarction or thrombosis, marked basal or perivillous fibrin deposition, increased syncytial knots) were inversely related to those associated with inflammation of the membranes and cord. Earlier age favoured inflammatory variables, while older age favoured characteristics attributed to impaired blood flow. We observed inflammation of the chorionic plate in 43%, the cord in 19%, and of chorionic plate vessels in 30%. Of the placentas with umbilical cord inflammation, 8% had no inflammation of the chorionic plate. Conclusions: This study population is unique in its size and recruitment by gestational age rather than birth weight. Inflammation occurred frequently, but not in placentas that had characteristics of vasculopathy. The prevalence of inflammation decreased with increasing gestational age, while vasculopathy increased. Funisitis need not be accompanied by chorionic inflammation.

Inflammatory bowel disease causes reversible suppression of osteoblast and chondrocyte function in mice

Decreased bone density and stature can occur in pediatric patients with inflammatory bowel disease (IBD). Little is known about how IBD broadly impacts the skeleton. To evaluate the influence of an acute episode of IBD on growing bone, 4-wk-old mice were administered 5% dextran sodium sulfate (DSS) for 5 days to induce colitis and their recovery was monitored. During active disease and early recovery, trabecular bone mineral density, bone volume, and thickness were decreased. Cortical bone thickness, outer perimeter, and density were also decreased, whereas inner perimeter and marrow area were increased. These changes appear to maintain bone strength since measures of moments of inertia were similar between DSS-treated and control mice. Histological (static and dynamic), serum, and RNA analyses indicate that a decrease in osteoblast maturation and function account for changes in bone density. Unlike some conditions of bone loss, marrow adiposity did not increase. Similar to reports in humans, bone length decreased and correlated with decreases in growth plate thickness and chondrocyte marker expression. During disease recovery, mice experienced a growth spurt that led to their achieving final body weights and bone length, density, and gene expression similar to healthy controls. Increased TNF-alpha and decreased IGF-I serum levels were observed with active disease and returned to normal with recovery. Changes in serum TNF-alpha (increased) and IGF-I (decreased) paralleled changes in bone parameters and returned to normal values with recovery, suggesting a potential role in the skeletal response.

Inflammation biomarkers in vaginal fluid and preterm delivery

STUDY QUESTION Which inflammation biomarkers detected in the vaginal fluid are most informative for identifying preterm delivery (PTD) risk? SUMMARY ANSWER Elevated interleukin (IL)-6 at mid-trimester was associated with increased odds of spontaneous PTD at <35 weeks and with PTD plus histologic chorioamnionitis (HCA), and had the greatest sensitivity for detecting these two PTD subtypes. WHAT IS KNOWN ALREADY Maternal and/or fetal inflammation play a role in some preterm deliveries, therefore inflammation biomarkers might help to identify women at greater risk. STUDY DESIGN, SIZE, DURATION We examined 1115 women from the Pregnancy Outcomes and Community Health Study, a cohort study conducted from September 1998 through June 2004, for whom data were available on mid-pregnancy inflammatory biomarkers. PARTICIPANTS/MATERIALS, SETTING, METHODS At enrollment at 16-27 weeks gestation, vaginal fluid samples were collected from a swab and 15 eluted biomarkers were measured using the Meso Scale Discovery multiplex electrochemiluminescence platform. Associations of biomarkers with PTD were examined, according to clinical circumstance, week at delivery and presence/absence of HCA. Weighted logistic regression was used to determine odds ratios (OR) and 95% confidence intervals (CI) adjusted for race. Sensitivity and specificity were compared between individual and multiple biomarkers, identified by a bootstrapping method. MAIN RESULTS AND THE ROLE OF CHANCE Elevated IL-6 (>75th percentile) displayed the strongest association with spontaneous PTD <35 weeks (OR 2.3; CI 1.3-4.0) and PTD with HCA (OR 2.8; CI 1.4-6.0). The sensitivity of IL-6 to detect spontaneous PTD <35 weeks or PTD with HCA was 0.43 and 0.51, respectively, while specificity was 0.74 and 0.75, respectively. IL-6 plus IL1β, IL-6r, tumor necrosis factor-alpha or granulocyte-macrophage colony-stimulating factor increased specificity (range 0.84-0.88), but decreased sensitivity (range 0.28-0.34) to detect both PTD subtypes. Results were similar when a combination of IL-6 and bacterial vaginosis (BV) was explored. Thus, the use of multiple biomarkers did not detect PTD subtypes with a greater sensitivity than IL-6 alone, and IL-6 is a specific but non-sensitive marker for the detection of spontaneous PTD. LIMITATIONS, REASONS FOR CAUTION Our ability to find small effect size associations between PTD and inflammation biomarkers (OR <2.0) might have been limited by the modest number of less common PTD subtypes in our population (e.g. spontaneous delivery <35 weeks, PTD accompanied by HCA) and by relatively higher variability for some cytokines, for example tumor necrosis factor-α, IL-12p70, IL-10 and granulocyte-macrophage colony-stimulating factor, that are less stable and commonly undetectable or detectable at low levels in human vaginal secretions. WIDER IMPLICATIONS OF THE FINDINGS Larger studies are needed to further explore a role of inflammation biomarkers in combination with other risk factors, including specific BV-associated organisms, for the prediction of PTD subtypes. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the National Institute of Child Health and Human Development, National Institute of Nursing, March of Dimes Foundation, Thrasher Research Foundation and Centers for Disease Control and Prevention. The authors have no conflicts of interest.

Maternal lipids at mid-pregnancy and the risk of preterm delivery

Objective. This study examined associations between maternal lipid levels at mid‐pregnancy and preterm delivery, medically indicated or spontaneous. Design: Prospective cohort study. Setting. Women were recruited from 52 clinics in five Michigan, USA communities (1998–2004). Population. Pregnant women were enrolled at 15–27 weeks’ gestation and followed to delivery (n=3019). Methods. A single blood sample was obtained at study enrollment. Blood lipids, i.e. total cholesterol (TC), high‐density lipoprotein (HDLc), low‐density lipoprotein (LDLc) cholesterol, and triglycerides (TG), were measured on a sub‐cohort (n=1309). Main outcome measures. There were 221 spontaneous, 100 medically indicated preterm deliveries and 988 term deliveries. Polytomous logistic regression models examined relationships among cholesterol levels (Low: <10th percentile, Referent: 10th–<70th percentile, High: ≥70th percentile), quartiles of TG (Referent: first quartile) and delivery outcome (Referent: term). Results. Odds of medically indicated preterm delivery were increased among women with low TC (adjusted odds ratio (aOR)=2.04, 95% confidence interval (CI): 1.12, 3.72), low HDLc (aOR=1.89, 95%CI: 1.04, 3.42) or low LDLc (aOR=1.96, 95%CI: 1.09, 3.54). Odds of spontaneous preterm delivery were increased among women with high TC (aOR=1.51, 95%CI: 1.06, 2.15), high LDLc (aOR=1.42, 95%CI: 0.99, 2.04) or high TG (aOR=1.90, 95%CI: 1.21, 2.97 and aOR=1.72, 95%CI: 1.06, 2.78 for third and fourth quartiles, respectively). Conclusions. Extremely low TC, HDLc, and LDLc were associated with a modest increase in risk of medically indicated preterm delivery, whereas high TC, LDLc and TG modestly increased the risk of spontaneous preterm delivery. Further research is needed to uncover explanations for these associations and to identify optimal ranges for maternal lipids.

Myeloid-specific GPCR kinase-2 negatively regulates NF-κB1p105-ERK pathway and limits endotoxemic shock in mice.

G‐protein‐coupled receptor kinase 2 (GRK2) is a member of a kinase family originally discovered for its role in the phosphorylation and desensitization of G‐protein‐coupled receptors. It is expressed in high levels in myeloid cells and its levels are altered in many inflammatory disorders including sepsis. To address the physiological role of myeloid cell‐specific GRK2 in inflammation, we generated mice bearing GRK2 deletion in myeloid cells (GRK2▵mye). GRK2▵mye mice exhibited exaggerated inflammatory cytokine/chemokine production, and organ injury in response to lipopolysaccharide (LPS, a TLR4 ligand) when compared to wild‐type littermates (GRK2fl/fl). Consistent with this, peritoneal macrophages from GRK2▵mye mice showed enhanced inflammatory cytokine levels when stimulated with LPS. Our results further identify TLR4‐induced NF‐κB1p105‐ERK pathway to be selectively regulated by GRK2. LPS‐induced activation of NF‐κB1p105‐MEK‐ERK pathway is significantly enhanced in the GRK2▵mye macrophages compared to GRK2fl/fl cells and importantly, inhibition of the p105 and ERK pathways in the GRK2▵mye macrophages, limits the enhanced production of LPS‐induced cytokines/chemokines. Taken together, our studies reveal previously undescribed negative regulatory role for GRK2 in TLR4‐induced p105‐ERK pathway as well as in the consequent inflammatory cytokine/chemokine production and endotoxemia in mice. J. Cell. Physiol. 226: 627–637, 2011.

Polymorphisms in thrombophilia and renin–angiotensin system pathways, preterm delivery, and evidence of placental hemorrhage

OBJECTIVE The purpose of this study was to analyze functional polymorphisms in candidate genes (methylenetetrahydrofolate reductase [MTHFR]677C>T, MTHFR1298A>C, factor 5 1691G>A [FVL], and angiotensinogen (AGT)-6G>A) in relation to a hypothesized placental hemorrhage pathway to preterm delivery (PTD). STUDY DESIGN We assessed maternal genotypes, pregnancy outcomes, and placental pathologic evidence among 560 white and 399 black women who were recruited at mid trimester into a prospective cohort study (1998-2004). Odds of dominant genotypes were calculated for PTDs with (n = 56) or without (n = 177) evidence of placental hemorrhage (referent = term) with the use of race-stratified polytomous logistic regression models. RESULTS Among white women, FVL GA/AA and AGT(-6) GA/AA were both associated with hemorrhage-related PTDs (odds ratio [OR], 4.8; 95% confidence interval [CI], 1.6-14.2 and OR, 3.8; 95% CI, 1.3-10.5, respectively), but not other PTDs (ORs, 1.2 and 0.9, respectively). FVL GA/AA was associated with placental abruption (OR, 5.8; 95% CI, 1.1-30) among white women. All results were null for MTHFR genotypes. CONCLUSION FVL and AGT variant genotypes were associated specifically with hemorrhage-related PTDs.

Maternal Catecholamine Levels in Midpregnancy and Risk of Preterm Delivery

Associations between stress hormones and preterm delivery have not been fully explored. In this study, pregnant women enrolled from 52 clinics in 5 Michigan communities (1998-2004) provided urine samples for 3 days (waking and bedtime) during midpregnancy. Urinary catecholamine levels (epinephrine, norepinephrine, and dopamine) were measured in a subcohort (247 preterm and 760 term deliveries), and a 3-day median value was calculated. Polytomous logistic regression models assessed relations between catecholamine quartiles (of the median) and a 4-level outcome variable (i.e., term (referent) and 3 preterm delivery subtypes: spontaneous; premature rupture of membranes; and medically indicated). Final models incorporated other relevant covariates (e.g., creatinine, demographic, behavior). The risk of spontaneous preterm delivery was increased in the highest versus lowest quartile of norepinephrine and dopamine: norepinephrine, waking (adjusted odds ratio (AOR) = 3.7, 95% confidence interval (CI): 1.8, 7.9) and bedtime (AOR = 2.5, 95% CI: 1.3, 4.9); dopamine, waking (AOR = 2.6, 95% CI: 1.4, 5.1) and bedtime (AOR = 2.3, 95% CI: 1.2, 4.6). Adjusted odds ratios were further strengthened after removing women whose placentas showed evidence of acute infection or vascular pathology. High catecholamine levels in maternal urine may be indicative of excess stressors and/or predisposition to elevated sympathetic activation that contributes to increased risk of spontaneous preterm delivery.

Downregulation of H19 Improves the Differentiation Potential of Mouse Parthenogenetic Embryonic Stem Cells

Parthenogenetic embryonic stem cells (P-ESCs) offer an alternative source of pluripotent cells, which hold great promise for autologous transplantation and regenerative medicine. P-ESCs have been successfully derived from blastocysts of several mammalian species. However, compared with biparental embryonic stem cells (B-ESCs), P-ESCs are limited in their ability to fully differentiate into all 3 germ layers. For example, it has been observed that there is a differentiation bias toward ectoderm derivatives at the expense of endoderm and mesoderm derivatives-muscle in particular-in chimeric embryos, teratomas, and embryoid bodies. In the present study we found that H19 expression was highly upregulated in P-ESCs with more than 6-fold overexpression compared with B-ESCs. Thus, we hypothesized that manipulation of the H19 gene in P-ESCs would alleviate their limitations and allow them to function like B-ESCs. To test this hypothesis we employed a small hairpin RNA approach to reduce the amount of H19 transcripts in mouse P-ESCs. We found that downregulation of H19 led to an increase of mesoderm-derived muscle and endoderm in P-ESCs teratomas similar to that observed in B-ESCs teratomas. This phenomenon coincided with upregulation of mesoderm-specific genes such as Myf5, Myf6, and MyoD. Moreover, H19 downregulated P-ESCs differentiated into a higher percentage of beating cardiomyocytes compared with control P-ESCs. Collectively, these results suggest that P-ESCs are amenable to molecular modifications that bring them functionally closer to true ESCs.

Association of maternal igm concentrations above the median at 15-19 weeks of gestation and early preterm delivery

IgM concentrations greater than the median in maternal serum collected at 15-19 weeks of pregnancy were strongly associated (odds ratio 15.6) with delivery before 29 weeks. Our results suggest that the postulated inflammatory component in the aetiology of very preterm delivery may elicit a measurable maternal humoral response as early as mid-trimester.