Jacob Larkin

Perinatal Outcomes and Unconventional Natural Gas Operations in Southwest Pennsylvania

Unconventional gas drilling (UGD) has enabled extraordinarily rapid growth in the extraction of natural gas. Despite frequently expressed public concern, human health studies have not kept pace. We investigated the association of proximity to UGD in the Marcellus Shale formation and perinatal outcomes in a retrospective cohort study of 15,451 live births in Southwest Pennsylvania from 2007–2010. Mothers were categorized into exposure quartiles based on inverse distance weighted (IDW) well count; least exposed mothers (first quartile) had an IDW well count less than 0.87 wells per mile, while the most exposed (fourth quartile) had 6.00 wells or greater per mile. Multivariate linear (birth weight) or logistical (small for gestational age (SGA) and prematurity) regression analyses, accounting for differences in maternal and child risk factors, were performed. There was no significant association of proximity and density of UGD with prematurity. Comparison of the most to least exposed, however, revealed lower birth weight (3323 ± 558 vs 3344 ± 544 g) and a higher incidence of SGA (6.5 vs 4.8%, respectively; odds ratio: 1.34; 95% confidence interval: 1.10–1.63). While the clinical significance of the differences in birth weight among the exposure groups is unclear, the present findings further emphasize the need for larger studies, in regio-specific fashion, with more precise characterization of exposure over an extended period of time to evaluate the potential public health significance of UGD.

A customized standard of large size for gestational age to predict intrapartum morbidity

OBJECTIVE The purpose of this study was to determine whether a customized standard of large-for-gestational age (LGA) identifies pregnancies with increased perinatal risk. STUDY DESIGN We evaluated 7510 estimates of fetal weight to generate a fetal growth curve. Next, we analyzed the gestational age at delivery, physiologic and pathological variables from 5072 pregnancies to predict birthweight, and calculated a customized ideal birthweight and cutoff for LGA. In a separate analysis of 32,271 pregnancies, rates of macrosomia-related adverse outcomes were compared in pregnancies that had been identified as LGA by a customized standard (LGA(cust)) and those pregnancies that had been identified as LGA or macrosomic by conventional standards. RESULTS LGA(cust) pregnancies carried increased risk of shoulder dystocia, third- or fourth-degree laceration, and cephalopelvic disproportion. LGA(cust) pregnancies that did not meet conventional criteria for LGA/macrosomia were at increased risk of all measured outcomes. CONCLUSION A customized standard of LGA identifies a previously unrecognized population that is at increased risk of perinatal morbidity.

Risk of morbid perinatal outcomes in small-for-gestational-age pregnancies: customized compared with conventional standards of fetal growth.

OBJECTIVE: To estimate and compare the risk of morbid perinatal outcomes in pregnancies identified as small for gestational age (SGA) with customized compared with conventional standards of fetal growth. METHODS: Ultrasound-derived estimates of fetal weight were used to generate a fetal growth trajectory (N=7,510). The gestational age at delivery and pathologic and physiologic variables from 5,072 pregnancies were used to calculate a customized threshold for SGA. In a separate analysis of 32,070 pregnancies, rates of morbid outcomes were compared in participants classified as SGA according to a population-based birth weight standard only (SGApop only), a customized standard only (SGAcust only), and both methods (SGAboth). RESULTS: Eight-hundred seventy-five (2.7%) participants were SGApop only, 1,970 (6.1%) participants were SGAboth, and 609 (1.9%) participants were SGAcust only. The odds ratios of neonatal death in SGApop only and SGAcust only pregnancies were 1.78 (95% confidence interval [CI] 0.2–13.1) and 54.6 (95% CI 29.0–102.8), respectively. Rates of prematurity in the SGApop only and SGAcust only cohorts were 4.8% and 64.5%, respectively. After adjustment for the effect of prematurity, odds ratios of neonatal death in the SGApop only and SGAcust only cohorts were 4.8 (95% CI 0.6–37.0) and 2.9 (95% CI 1.4–6.1), respectively. CONCLUSION: After adjustment for confounding stemming from premature delivery, there is little difference in the risk of adverse outcomes between SGAcust only and SGApop only participants. Adoption of customized fetal growth standards into clinical practice may not improve the ability to identify pregnancies with increased risk of perinatal morbidity. LEVEL OF EVIDENCE: II

Neonatal outcomes following preterm birth classified according to placental features

BACKGROUND: Preterm birth has staggering health implications, and yet the causes of most cases are still unknown. Placental features have been understudied as an etiology for preterm birth, and the association between placental pathologic lesions and neonatal outcomes are incompletely understood. OBJECTIVE: We sought to characterize births according to placental pathology and relate these to adverse neonatal outcomes. STUDY DESIGN: We studied 20,091 births (15,710 term and 4381 preterm) with placental evaluations. Births were classified according to the presence or absence of placental lesions consistent with malperfusion (vasculopathy, infarct, advanced villous maturation, perivillous fibrin, fibrin deposition) and intrauterine inflammation/infection (chorioamnionitis, funisitis, vasculitis). Outcomes were gestational week of delivery, birthweight z‐score, neonatal respiratory distress syndrome, and intraventricular hemorrhage. RESULTS: Among all preterm births, evidence of placental malperfusion was identified more often than inflammation/infection (50.6% vs 27.3%, P < .0001). Placental malperfusion was associated with reduced fetal growth (adjusted birthweight z‐score, –0.83, P < .0001) and lesions of inflammation/infection were associated with earlier delivery (adjusted difference –2.08 weeks, P < .0001) than those with no lesions. When both placental lesions were present, earlier delivery (adjusted difference –2.28 weeks, P < .0001) and reduced fetal growth (adjusted birthweight z‐score difference, –0.24, P = .001) were observed more often than when neither lesion was present. Findings were similar when restricted to cases of spontaneous preterm birth. Intraventricular hemorrhage was higher in preterm births with malperfusion lesions than cases with no lesions (7.6% vs 3.4%; odds ratio, 1.98; confidence interval, 1.18–3.32), accounting for gestational age and other covariates. CONCLUSION: Placental pathology provides important insight into subtypes of preterm birth with adverse neonatal outcomes. Co‐occurrence of malperfusion and inflammation/infection, especially among spontaneous preterm births, may be a novel pattern of placental injury linked to severe adverse outcomes.

The Expression and Localization of N-Myc Downstream-Regulated Gene 1 in Human Trophoblasts

The protein N-Myc downstream-regulated gene 1 (NDRG1) is implicated in the regulation of cell proliferation, differentiation, and cellular stress response. NDRG1 is expressed in primary human trophoblasts, where it promotes cell viability and resistance to hypoxic injury. The mechanism of action of NDRG1 remains unknown. To gain further insight into the intracellular action of NDRG1, we analyzed the expression pattern and cellular localization of endogenous NDRG1 and transfected Myc-tagged NDRG1 in human trophoblasts exposed to diverse injuries. In standard conditions, NDRG1 was diffusely expressed in the cytoplasm at a low level. Hypoxia or the hypoxia mimetic cobalt chloride, but not serum deprivation, ultraviolet (UV) light, or ionizing radiation, induced the expression of NDRG1 in human trophoblasts and the redistribution of NDRG1 into the nucleus and cytoplasmic membranes associated with the endoplasmic reticulum (ER) and microtubules. Mutation of the phosphopantetheine attachment site (PPAS) within NDRG1 abrogated this pattern of redistribution. Our results shed new light on the impact of cell injury on NDRG1 expression patterns, and suggest that the PPAS domain plays a key role in NDRG1’s subcellular distribution.

The use of angiogenic factors in discriminating preeclampsia: are they ready for prime time?

Objectives. We sought to evaluate the association between soluble fms-like tyrosine kinase 1 (sFlt1) and endoglin (ENG) and preeclampsia in an urban population, to develop a discriminatory model, and evaluate the association of these biomarkers with small for gestational age (SGA). Methods. Cases are prospectively identified with preeclampsia. Controls are term patients without preeclampsia. Commercially available ELISAs were used to measure levels of sFlt1, ENG, and placental growth factor (PlGF). Log-transformed levels were compared and multivariable logistic regression analyses were performed to control for confounders. Receiver operating characteristic curves were developed. Results. In cases (n = 86) compared to controls (n = 288), sFlt1 (p = 0.24) levels were no different. However, ENG levels were higher (p < 0.001), and PlGF levels were lower (p < 0.001). Further, levels of sFlt1 had poor discriminatory ability between cases and controls [AUC = 0.56, (0.48–0.63)]. The best model to discriminate between groups included clinical risk factors, ENG, and PlGF [AUC = 0.89, (0.85–0.92)]. Conclusions. Unlike recent reports, this study suggests that sFlt1 may have limited diagnostic utility in predicting preeclampsia, especially term disease.

The influence of ligand-activated LXR on primary human trophoblasts

INTRODUCTION The Liver X Receptors (LXRs) are critical transcriptional regulators of cellular metabolism that promote cholesterol efflux and lipogenesis in response to excess intracellular cholesterol. In contrast, the Sterol Response Element Binding Protein-2 (SREBP2) promotes the synthesis and uptake of cholesterol. Oxysterols are products of cholesterol oxidation that accumulate in conditions associated with increased cellular levels of reactive oxygen species, such as hypoxia and oxidative stress, activating LXR and inhibiting SREBP2. While hypoxia and oxidative stress are commonly implicated in placental injury, the impact of the transcriptional regulation of cholesterol homeostasis on placental function is not well characterized. METHODS We measured the effects of the synthetic LXR ligand T0901317 and the endogenous oxysterol 25-hydroxycholesterol (25OHC) on differentiation, cytotoxicity, progesterone synthesis, lipid droplet formation, and gene expression in primary human trophoblasts. RESULTS Exposure to T0901317 promoted lipid droplet formation and inhibited differentiation, while 25OHC induced trophoblast toxicity, promoted hCG and progesterone release at lower concentrations with inhibition at higher concentrations, and had no effect on lipid droplet formation. The discrepant effect of these ligands was associated with distinct changes in expression of LXR and SREBP2 target genes, with upregulation of ABCA1 following 25OHC and T090317 exposure, exclusive activation of the lipogenic LXR targets SREBP1c, ACC1 and FAS by T0901317, and exclusive inhibition of the SREBP2 targets LDLR and HMGCR by 25OHC. CONCLUSION These findings implicate cholesterol oxidation as a determinant of trophoblast function and activity, and suggest that placental gene targets and functional pathways are selectively regulated by specific LXR ligands.

NDRG1 Deficiency Attenuates Fetal Growth and the Intrauterine Response to Hypoxic Injury

Intrauterine mammalian development depends on the preservation of placental function. The expression of the protein N-myc downstream-regulated gene 1 (NDRG1) is increased in placentas of human pregnancies affected by fetal growth restriction and in hypoxic primary human trophoblasts, where NDRG1 attenuates cell injury. We sought to assess the function of placental NDRG1 in vivo and tested the hypothesis that NDRG1 deficiency in the mouse embryo impairs placental function and consequently intrauterine growth. We found that Ndrg1 knock-out embryos were growth restricted in comparison to wild-type or heterozygous counterparts. Furthermore, hypoxia reduced the survival of female, but not male, knock-out embryos. Ndrg1 deletion caused significant alterations in placental gene expression, with a marked reduction in transcription of several lipoproteins in the placental labyrinth. These transcriptional changes were associated with reduced fetal:maternal serum cholesterol ratio exclusively in hypoxic female embryos. Collectively, our findings indicate that NDRG1 promotes fetal growth and regulates the metabolic response to intrauterine hypoxic injury in a sexually dichotomous manner.

Small for Gestational Age: The Differential Mortality When Detected versus Undetected Antenatally.

Background Antenatal detection of fetal growth restriction (FGR) prompts antepartum surveillance to reduce perinatal mortality, yet most cases of FGR are undetected. Objective This study aims to compare rates of adverse neonatal outcomes when FGR is detected versus undetected. Study Design Small‐for‐gestational‐age (SGA) newborns (birth weight < 10% for gestational age) delivered at the Magee‐Womens Hospital in Pittsburgh, PA from 2003 to 2010 were divided into three groups: those whom did not undergo third‐trimester fetal growth ultrasound (SGA‐no US), were appropriate for gestational age (AGA) by ultrasound (SGA‐undetected), or were FGR by ultrasound (SGA‐detected). We then compared rates of 5‐minute Apgar < 4 and neonatal death (ND), with AGA newborns as the referent. Results Out of 29,885 neonates, 2,475 (8.3%) were SGA. Out of the 826 (33%) SGA neonates who underwent growth ultrasound, 185 (22%) were considered FGR. In the SGA‐no US group, the adjusted odds ratio (aOR) for Apgar < 4 was 2.84 (95% confidence interval (CI): 1.28‐6.29) and 3.87 (95% CI: 2.09‐7.18) for ND. The risk of Apgar < 4 (aOR: 3.10, 95% CI: 0.93‐10.28) and ND (aOR: 2.16, 95% CI: 0.66‐7.14) were not significantly elevated for SGA‐undetected neonates, while SGA‐detected neonates were most at risk, with an aOR of 18.20 (95% CI: 6.82‐48.60) for Apgar < 4 and 18.24 (95% CI: 7.90‐42.13) for ND. Conclusion Fetal growth ultrasound effectively stratifies risk amongst SGA neonates.

Impairment of trophoblast survival and differentiation by LXR ligands is prevented by cholesterol but not ABCA1 silencing

INTRODUCTION The Liver X Receptors (LXRs) drive the transcriptional response to excess intracellular cholesterol. Oxysterols, the products of cholesterol oxidation, are activating ligands for LXR that can accumulate under conditions of oxidative stress and disrupt cholesterol homeostasis. While activation of LXR inhibits trophoblast differentiation, the impact of LXR on trophoblast physiology or cholesterol homeostasis is incompletely understood. We sought to determine if the effects of LXR activation can be ameliorated through modification of cholesterol bioavailability or inhibition of LXR-driven cholesterol efflux in trophoblasts. METHODS We measured the effect of oxysterol exposure on BeWo cells and primary human trophoblasts (PHT cells) cultured in lipoprotein-deficient medium. We also measured the effect of the synthetic, LXR-specific ligand T0901317 on PHT cell differentiation and survival. Finally, we silenced the ATP-binding cassette transporter A1 (ABCA1), a transcriptional target of LXR that drives cholesterol efflux, to determine if inhibition of cholesterol efflux could block the effects of T0901317. RESULTS Oxysterols inhibited BeWo survival and PHT cell differentiation, and these effects were blocked by cholesterol supplementation. T0901317 also inhibited PHT cell differentiation, and this effect was similarly blocked by cholesterol. Unlike cholesterol however, ABCA1 silencing did not modify the effect of T0901317 on PHT cell differentiation. DISCUSSION Oxysterols and LXR inhibit trophoblast survival and differentiation exclusively in conditions of cholesterol scarcity. These findings underscore the importance of cholesterol homeostasis in the maintenance of placental function and suggest that pathways regulating cholesterol homeostasis may represent therapeutic targets to mitigate harmful sequelae of placental injury.