Code Status Blues: Do Legal Nudges Discourage Doctors From Ordering Do-Not-Resuscitate?

BackgroundLaws influence human behavior, including practitioners' behavior, and legal nudges may affect bedside patient care practices. Do-not-resuscitate (DNR) practices are one such example. Ensuring that practitioners order DNR for patients who request it is a crucial part of providing quality end-of-life care. On April 1, 2018, in the state of Texas, Senate Bill 11 (SB 11) took effect. This law did not make DNR orders illegal, but it constrained and complicated the process for issuing them. This study aimed to determine if DNR order utilization decreased after the law's implementation. MethodsThe authors conducted a retrospective cohort chart review of all adult patients admitted to a single academic urban tertiary care hospital in Texas before and after the state's DNR law went into effect. The authors reviewed code status orders for the 5426 sickest patients. The primary outcome is the proportion of patients who had DNR orders in effect at the end of their hospitalizations. ResultsImplementation of the DNR law's cumbersome documentation and witnessing requirements correlated with a substantial decline in DNR orders for patients at the highest risk of dying from chronic or severe illness. ConclusionThis is the first study the authors know of that examines whether DNR usage declined after implementation of a DNR law. A troubling implication of this study is that the Texas law has had a chilling effect on doctors' willingness and ability to place medically and ethically appropriate DNR orders and has threatened the right of patients with serious illness to forgo cardiopulmonary resuscitation.

Developing Health Literacy Communication Practices for Medical Students.

Introduction: Health literacy and its associated communication practices are critical to patient-centered care and have been endorsed by various associations as important for health professional training. Unfortunately, there is little published literature on how to teach health literacy to medical students and health professionals. Methods: We developed a two-part curriculum during a required module for medical students including an introductory session in their first year and a skill-building workshop in their second year. In the workshop, students studied, observed, and practiced three health literacy communication techniques: teach-back, avoiding jargon, and effective questioning. Results: The workshop was implemented with approximately 100 second-year medical students as part of a course in their required curriculum. Results of a Wilcoxon rank sum test of pre/post survey responses showed a statistically significant move towards conviction of importance and confidence in ability to use three health literacy techniques. Discussion: A skills-based workshop on health literacy skills can improve medical students' conviction and confidence in using health literacy communication practices.

Increased oxidative stress in patients with depression and its relationship to treatment.

Oxidative stress may play a role in the pathogenesis of depression. We tested the hypothesis that urinary F2 isoprostanes, a robust marker of oxidative stress, was increased in patients with depression and associated with symptoms and response to treatment. Urinary F2 isoprostanes was compared in 18 patients with depression and 36 age and sex matched control subjects. In patients, we tested the association between oxidative stress, depression questionnaires and antidepressant treatment. Urinary F2 isoprostane excretion was significantly higher in patients with depression than in control subjects. This association remained significant after adjustment for age, sex and BMI. Depression symptom severity scores were not correlated with F2 isoprostane excretion. Nine patients were treated with sertraline or bupropion for 8 weeks. Depression severity rating scale scores decreased significantly and F2 isoprostane excretion increased. The increase in F2 isoprostane excretion was inversely correlated with the improvement in Hamilton Depression Rating 17 items. In conclusion, oxidative stress is increased in patients with depression. However, although treatment with either bupropion or sertraline reduces the symptoms of depression, it may increase F2 isoprostane excretion. These results suggest that alternative mechanisms, beyond oxidative stress, may be involved in the development of depression and subsequent responses to treatment.

Sex differences in urinary biomarkers of vascular and endothelial function in HIV-infected persons receiving antiretroviral therapy.

BACKGROUND: Cardiovascular disease (CVD) risk can be underestimated in HIV-infected patients receiving antiretroviral therapy (ART). Novel CVD risk markers in this population are needed. We hypothesized that eicosanoid metabolite production is increased with metabolic complications of ART. Our objective was to determine relationships between urine eicosanoids and traditional CVD risk factors in a cohort of HIV-infected persons receiving ART. METHODS: Cross-sectional analysis of 107 individuals from a prospective cohort study with urine eicosanoids (isoprostane [15-F(2t)-IsoP], prostaglandin-E metabolite [PGE-M], thromboxane metabolite [11dTxB(2)], prostacyclin metabolite [PGI-M]) determined by gas or liquid chromatography-mass spectrometry. RESULTS: 15-F(2t)-IsoP was higher (P=0.003), 11dTxB(2) tended to be higher (P=0.07) and PGE-M was lower (P=0.003) in females than in males. The overall median Framingham score was 4 (IQR 1-7). In multivariable analyses adjusting for age, CD4(+) T-cells, smoking status, non-steroidal anti-inflammatory drug use, aspirin use and body mass index (BMI), associations included: higher 15-F(2t)-IsoP with female sex (P=0.004) and current smoking (P=0.04), lower PGE-M with female sex (P=0.005) and higher BMI (P=0.03), higher 11dTxB(2) with increasing age (P=0.02) and current smoking (P=0.04), lower 11dTxB(2) with higher BMI (P=0.02), and higher PGI-M with current smoking (P=0.04). CONCLUSIONS: In this pilot study of predominantly virologically suppressed HIV-infected individuals on ART, there were sex-specific differences in urinary eicosanoids, with females having more risk-associated parameters despite a low Framingham score. Eicosanoids might be useful CVD biomarkers in ART-treated, HIV-infected patients. Future studies should examine eicosanoids while assessing effects of specific ART regimens and targeted interventions on CVD outcomes.

The fatty acid oxidation product 15-A3t-isoprostane is a potent inhibitor of NFκB transcription and macrophage transformation.

Fatty acids such as eicosapentaenoic acid (EPA) have been shown to be beneficial for neurological function and human health. It is widely thought that oxidation products of EPA are responsible for biological activity, although the specific EPA peroxidation product(s) which exert these responses have not yet been identified. In this work we provide the first evidence that the synthesized representative cyclopentenone IsoP, 15-A(3t)-IsoP, serves as a potent inhibitor of lipopolysaccharide-stimulated macrophage activation. The anti-inflammatory activities of 15-A(3t)-IsoP were observed in response not only to lipopolysaccharide, but also to tumor necrosis factor alpha and IL-1b stimulation. Subsequently, this response blocked the ability of these compounds to stimulate nuclear factor kappa b (NFκB) activation and production of proinflammatory cytokines. The bioactivity of 15-A(3t)-IsoP was shown to be dependent upon an unsaturated carbonyl residue which transiently adducts to free thiols. Site directed mutagenesis of the redox sensitive C179 site of the Ikappa kinase beta subunit, blocked the biological activity of 15-A(3t)-IsoP and NFκB activation. The vasoprotective potential of 15-A(3t)-IsoP was underscored by the ability of this compound to block oxidized lipid accumulation, a critical step in foam cell transformation and atherosclerotic plaque formation. Taken together, these are the first data identifying the biological activity of a specific product of EPA peroxidation, which is formed in abundance in vivo. The clear mechanism linking 15-A(3t)-IsoP to redox control of NFκB transcription, and the compound's ability to block foam cell transformation suggest that 15-A(3t)-IsoP provides a unique and potent tool to provide vaso- and cytoprotection under conditions of oxidative stress.

PGI synthase overexpression protects against bleomycin-induced mortality and is associated with increased Nqo 1 expression.

The mortality rate for acute lung injury (ALI) is reported to be between 35-40%, and there are very few treatment strategies that improve the death rate from this condition. Previous studies have suggested that signaling through the prostaglandin (PG) I(2) receptor may protect against bleomycin-induced ALI in mice. We found that mice that overexpress PGI synthase (PGIS) in the airway epithelium were significantly protected against bleomycin-induced mortality and had reduced parenchymal consolidation, apoptosis of lung tissue, and generation of F(2)-isoprostanes compared with littermate wild-type controls. In addition, we show for the first time in both in vivo and in vitro experiments that PGI(2) induced the expression of NADP (H): quinoneoxidoreductase 1 (Nqo 1), an enzyme that prevents the generation of reactive oxygen species. PGI(2) induction of Nqo 1 provides a possible novel mechanism by which this prostanoid protects against bleomycin-induced mortality and identifies a potential therapeutic target for human ALI.

Oxidative stress measured by urine F2-isoprostane level is associated with prostate cancer.

PURPOSE: Oxidative stress is implicated in prostate cancer by several lines of evidence. We studied the relationship between the level of F2-isoprostanes, a validated biomarker of oxidative stress, and prostate cancer and high grade prostatic intraepithelial neoplasia. MATERIALS AND METHODS: This case-control analysis within the Nashville Men's Health Study included men recruited at prostate biopsy. Body morphometrics, health history and urine were collected from more than 2,000 men before biopsy. F2-isoprostanes were measured by gas chromatography/mass spectrometry within an age matched sample of Nashville Men's Health Study participants that included 140 patients with high grade prostatic intraepithelial neoplasia, 160 biopsy negative controls and 200 prostate cancer cases. Multivariable linear and logistic regression was used to determine the associations between F2-isoprostane level, and high grade prostatic intraepithelial neoplasia and prostate cancer. RESULTS: Mean patient age was 66.9 years (SD 7.2) and 10.1% were nonwhite. Adjusted geometric mean F2-isoprostane levels were higher in patients with prostate cancer (1.82, 95% CI 1.66-2.00) or high grade prostatic intraepithelial neoplasia (1.82, 95% CI 1.68-1.96) than in controls (1.63, 95% CI 1.49-1.78, p <0.001), but were similar across Gleason scores (p = 0.511). The adjusted odds of high grade prostatic intraepithelial neoplasia and prostate cancer increased with increasing F2-isoprostane quartile (p-trend = 0.015 and 0.047, respectively) and the highest F2-isoprostane quartile was associated with significantly increased odds of prostate cancer (OR 2.44, 95% CI 1.17-5.09, p = 0.017). CONCLUSIONS: Pre-diagnosis urine F2-isoprostane level is increased in men with high grade prostatic intraepithelial neoplasia or prostate cancer, suggesting urinary F2-isoprostane provides a biomarker for the role for oxidative stress in prostate carcinogenesis. F2-isoprostanes may also serve to estimate the efficacy of interventions targeting oxidative stress mechanisms in prostate cancer prevention or treatment.

p66(shc)'s role as an essential mitophagic molecule in controlling neuronal redox and energetic tone.

Stroke is the leading cause of adult disability in the U.S. and is now recognized as a global epidemic. There are currently no FDA-approved drugs to block the cell death that results from oxygen and glucose deprivation. This void in clinical medicine has sparked an intense interest in understanding endogenous cellular protective pathways that might be exploited for therapeutic development. The work highlighted here describes the critical role between redox tone and energetic stress signaling in mediating mitophagy and determining neuronal cell fate following acute oxygen glucose deprivation.

Essential role of the redox-sensitive kinase p66shc in determining energetic and oxidative status and cell fate in neuronal preconditioning.

Ischemic preconditioning is a phenomenon in which low-level stressful stimuli upregulate endogenous defensive programs, resulting in subsequent resistance to otherwise lethal injuries. We previously observed that signal transduction systems typically associated with neurodegeneration such as caspase activation are requisite events for the expression of tolerance and induction of HSP70. In this work, we sought to determine the extent and duration of oxidative and energetic dysfunction as well as the role of effector kinases on metabolic function in preconditioned cells. Using an in vitro neuronal culture model, we observed a robust increase in Raf and p66(Shc) activation within 1 h of preconditioning. Total ATP content decreased by 25% 3 h after preconditioning but returned to baseline by 24 h. Use of a free radical spin trap or p66(shc) inhibitor increased ATP content whereas a Raf inhibitor had no effect. Phosphorylated p66(shc) rapidly relocalized to the mitochondria and in the absence of activated p66(shc), autophagic processing increased. The constitutively expressed chaperone HSC70 relocalized to autophagosomes. Preconditioned cells experience significant total oxidative stress measured by F(2)-isoprostanes and neuronal stress evaluated by F(4)-neuroprostane measurement. Neuroprostane levels were enhanced in the presence of Shc inhibitors. Finally, we found that inhibiting either p66(shc) or Raf blocked neuroprotection afforded by preconditioning as well as upregulation of HSP70, suggesting both kinases are critical for preconditioning but function in fundamentally different ways. This is the first work to demonstrate the essential role of p66(shc) in mediating requisite mitochondrial and energetic compensation after preconditioning and suggests a mechanism by which protein and organelle damage mediated by ROS can increase HSP70.

Cyclosporine A suppresses keratinocyte cell death through MPTP inhibition in a model for skin cancer in organ transplant recipients.

Transplant recipients have an elevated risk of skin cancer, with a 65- to 250-fold increase in squamous cell carcinoma. Usage of the immunosuppressant cyclosporine A (CsA) is associated with the development of skin cancer. We hypothesized that the increased incidence of skin cancer was due to the action of CsA within keratinocyte mitochondria where it can inhibit mitochondrial permeability transition pore (MPTP) opening. Normally, MPTP opening is induced by oxidative stress such as that caused by UV light and leads to cell death, thereby eliminating a cell that has been exposed to genotoxic insult. However, in the presence of CsA, damaged cells may survive and consequently form tumors. To test this hypothesis, we treated keratinocytes with levels of CsA used therapeutically in transplant patients and assessed their viability following UVA-irradiation. CsA prevented cell death by inhibiting MPTP opening, even though the levels of oxidative stress were increased markedly. Nim811, a non-immunosuppressive drug that can block the MPTP had a similar effect while the immunosuppressive drug tacrolimus that does not interact with the mitochondria had no effect. These findings suggest that CsA may promote skin cancer in transplant patients by allowing keratinocyte survival under conditions of increased genotoxic stress.

Comparison of three oxidative stress biomarkers in a sample of healthy adults.

Oxidative stress is a potentially important aetiological factor for many chronic diseases, including cardiovascular disease, neurodegenerative disease and cancer, yet studies often find inconsistent results. The associations between three of the most widely used biomarkers of oxidative stress, i.e. F(2)-isoprostanes for lipid peroxidation and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and the comet assay with FPG for oxidative DNA damage, were compared in a sample of 135 healthy African-American and white adults. Modest associations were observed between F(2)-isoprostanes and the comet assay (r = 0.22, p = 0.01), but there were no significant correlations between 8-oxo-dG and the comet assay (r = -0.09) or F(2)-IsoP (r = -0.04). These results are informative for researchers seeking to compare results pertaining to oxidative stress across studies and/or assessment methods in healthy disease-free populations. The development and use of oxidative stress biomarkers is a promising field; however, additional validation studies are necessary to establish accuracy and comparability across oxidative stress biomarkers.

Highly sensitive C-reactive protein, body mass index, and serum lipids in HIV-infected persons receiving antiretroviral therapy: a longitudinal study.

BACKGROUND: Antiretroviral therapy (ART) affects cardiovascular disease (CVD) risk. In the general population, highly sensitive creactive protein (hsCRP) is an established predictor of future coronary events. Little is known about its utility in chronic inflammatory conditions such as HIV infection. We assessed relationships between hsCRP and metabolic parameters over time in HIV-infected patients on ART. METHODS: Data are from a prospective cohort of HIV-infected adults enrolled June 2005 to July 2007. Participants were receiving ART, had HIV-1 RNA,10,000 copies per milliliter, and no diabetes or CVD. Nonlinear mixed-effect regression models assessed relationships between body mass index (BMI), lipids, and hsCRP over time adjusting for covariates. RESULTS: Ninety-four individuals had data from $1 study visit. Median age was 44 years, 27% were female, 57% white, and 54% were on protease inhibitors. Median CD4+ T cells, HIV-1 RNA, and hsCRP were 502 cells per cubic millimeter, 50 copies per milliliter, and 2.94 mg/dL, respectively. Median Framingham score was 3. Multivariate analysis identified associations between increased hsCRP and greater BMI (P = 0.001), higher non-high-density lipoprotein cholesterol (P = 0.013) and triglycerides (P = 0.017), and lower high-density lipoprotein cholesterol (P = 0.015). CONCLUSIONS: Among HIV-infected adults with low estimated CVD risk and virologic suppression on ART, hsCRP was elevated and independently associated with BMI and lipid changes. Future studies should assess associations between hsCRP and clinical outcomes.

Dietary fish oil exerts hypolipidemic effects in lean and insulin sensitizing effects in obese LDLR-/- mice.

Obesity is often associated with dyslipidemia, insulin resistance, and hypertension. Together, these metabolic perturbations greatly increase the risk of developing cardiovascular disease and diabetes. Although fish oil is a well-established hypolipidemic agent, the mechanisms by which it mediates its lipid-lowering effects are not clear. In addition, it has not been established whether dietary fish oil has different effects in lean and obese mice. LDL receptor deficient (LDLR-/-) and leptin deficient mice on a LDLR-/- background (ob/ob;LDLR-/-) were fed a high fat diet (39% total fat) supplemented with 6% olive oil or fish oil for 6 wk. Fish oil supplementation resulted in lower concentrations of plasma total cholesterol (P < 0.01), triglycerides (P < 0.01), and free fatty acids (P < 0.001) in lean LDLR-/- mice, but not in ob/ob;LDLR-/- mice. In contrast, a fish oil diet did not modulate insulin sensitivity in lean LDLR-/- mice, but it improved insulin sensitivity in ob/ob;LDLR-/- mice (P < 0.05) compared with olive oil fed ob/ob;LDLR-/- mice. Interestingly, plasma adiponectin concentrations were significantly higher and hepatic steatosis was reduced in both mouse models upon fish oil feeding. Finally, fish oil fed LDLR-/- mice exhibited higher hepatic AMP activated protein kinase (AMPK) phosphorylation (P < 0.05), whereas AMPK phosphorylation was not elevated by fish oil feeding in ob/ob;LDLR-/- mice. Taken together, our data suggest that fish oil reduces hepatic steatosis in both lean and obese mice, has potent plasma lipid lowering effects in lean mice, and exerts insulin sensitizing effects in obese mice.

Oxidant stress in HIV-infected women from the Women's Interagency HIV Study.

BACKGROUND: Oxidant stress contributes to the pathogenesis of multiple conditions and can be assessed by measuring plasma F(2)-isoprostane concentrations. We hypothesized that oxidant stress is associated with plasma homocysteine concentration and risk factors for atherosclerosis in HIV-infected women. METHODS: We measured plasma F(2)-isoprostane concentrations in a cross-sectional study of 249 HIV-infected women attending the Bronx (NY, USA) site of the Women's Interagency HIV Study and assessed associations with plasma homocysteine concentration and other metabolic parameters by linear regression. RESULTS: In multivariate analysis, hepatitis C virus (HCV) viraemia, waist circumference, homocysteine concentration and serum aspartate aminotransferase level were positively associated with log F(2)-isoprostane concentration (all P<0.005). There was a trend for an inverse association between log F(2)-isoprostane and CD4(+) T-cell percentage (P=0.06). Among women with HCV infection, the FIB-4 index, an indirect marker of liver fibrosis derived from routine laboratory tests, was positively associated with log F(2)-isoprostane concentration. CONCLUSIONS: In this cross-sectional study of HIV-infected women, plasma F(2)-isoprostane concentration was positively associated with homocysteine concentration, as well as HCV infection, abdominal obesity and aspartate aminotransferase level.

Elevated levels of urinary prostaglandin e metabolite indicate a poor prognosis in ever smoker head and neck squamous cell carcinoma patients.

Cyclooxygenase (COX)-derived prostaglandin E(2) (PGE(2)) plays a role in the development and progression of several tumor types including head and neck squamous cell carcinoma (HNSCC). Measurements of urinary PGE metabolite (PGE-M) can be used as an index of systemic PGE(2) production. In ever smokers, increased levels of urinary PGE-M reflect increased COX-2 activity. In this study, we determined whether baseline levels of urinary PGE-M were prognostic for ever smoker HNSCC patients. A retrospective chart review of ever smoker HNSCC patients treated with curative intent was done. Fifteen of 31 evaluable patients developed progressive disease (recurrence or a second primary tumor) after a median follow-up of 38 months. There were no statistically significant differences between patients with (n = 15) or without disease progression (n = 16) with regard to stage, site, treatment received, smoking status, and aspirin use during follow-up. Median urinary PGE-M levels were significantly higher in HNSCC patients with disease progression (21.7 ng/mg creatinine) compared with patients without (13.35 ng/mg creatinine; P = 0.03). Importantly, patients with high baseline levels of urinary PGE-M had a significantly greater risk of disease progression (hazard ratio, 4.76, 95% CI, 1.31-17.30; P < 0.01) and death (hazard ratio, 9.54; 95% CI, 1.17-77.7; P = 0.01) than patients with low baseline levels of urinary PGE-M. These differences were most evident among patients with early-stage disease. Taken together, our findings suggest that high baseline levels of urinary PGE-M indicate a poor prognosis in HNSCC patients. Possibly, HNSCC patients with high COX-2 activity manifested by elevated urinary PGE-M will benefit from treatment with a COX-2 inhibitor.

Two pathways for cyclooxygenase-2 protein degradation in vivo.

COX-2, formally known as prostaglandin endoperoxide H synthase-2 (PGHS-2), catalyzes the committed step in prostaglandin biosynthesis. COX-2 is induced during inflammation and is overexpressed in colon cancer. In vitro, an 18-amino acid segment, residues 595-612, immediately upstream of the C-terminal endoplasmic reticulum targeting sequence is required for N-glycosylation of Asn(594), which permits COX-2 protein to enter the endoplasmic reticulum-associated protein degradation system. To determine the importance of this COX-2 degradation pathway in vivo, we engineered a del595-612 PGHS-2 (Delta 18 COX-2) knock-in mouse lacking this 18-amino acid segment. Delta 18 COX-2 knock-in mice do not exhibit the renal or reproductive abnormalities of COX-2 null mice. Delta 18 COX-2 mice do have elevated urinary prostaglandin E(2) metabolite levels and display a more pronounced and prolonged bacterial endotoxin-induced febrile response than wild type (WT) mice. Normal brain tissue, cultured resident peritoneal macrophages, and cultured skin fibroblasts from Delta 18 COX-2 mice overexpress Delta 18 COX-2 relative to WT COX-2 expression in control mice. These results indicate that COX-2 can be degraded via the endoplasmic reticulum-associated protein degradation pathway in vivo. Treatment of cultured cells from WT or Delta 18 COX-2 mice with flurbiprofen, which blocks substrate-dependent degradation, attenuates COX-2 degradation, and treatment of normal mice with ibuprofen increases the levels of COX-2 in brain tissue. Thus, substrate turnover-dependent COX-2 degradation appears to contribute to COX-2 degradation in vivo. Curiously, WT and Delta 18 COX-2 protein levels are similar in kidneys and spleens from WT and Delta 18 COX-2 mice. There must be compensatory mechanisms to maintain constant COX-2 levels in these tissues.

Neurotoxic lipid peroxidation species formed by ischemic stroke increase injury.

Stroke is the third leading cause of death in the United States, yet no neuroprotective agents for treatment are clinically available. There is a pressing need to understand the signaling molecules that mediate ischemic cell death and identify novel neuroprotective targets. Cyclopentenone isoprostanes (IsoPs), formed after free radical-mediated peroxidation of arachidonic acid, are used as markers of stress, but their bioactivity is poorly understood. We have recently shown that 15-A(2t)-IsoP is a potent neurotoxin in vitro and increases the free radical burden in neurons. In this work, we demonstrate that 15-A(2t)-IsoP is abundantly produced in stroke-infarcted human cortical tissue. Using primary neuronal cultures we found that minimally toxic exposure to 15-A(2t)-IsoP does not alter ATP content, but in combination with oxygen glucose deprivation resulted in a significant hyperpolarization of the mitochondrial membrane and dramatically increased neuronal cell death. In the presence of Ca(2+), 15-A(2t)-IsoP led to a rapid induction of the permeability transition pore and release of cytochrome c. Taken with our previous work, these data support a model in which ischemia causes generation of reactive oxygen species, calcium influx, lipid peroxidation, and 15-A(2t)-IsoP formation. These factors combine to enhance opening of the permeability transition pore leading to cell death subsequent to mitochondrial cytochrome c release. These data are the first documentation of significant 15-A(2t)-IsoP formation after acute ischemic stroke and suggest that the addition of 15-A(2t)-IsoP to in vitro models of ischemia may help to more fully recapitulate stroke injury.

Restoration of on-time embryo implantation corrects the timing of parturition in cytosolic phospholipase A2 group IVA deficient mice.

Cytosolic phospholipase A2 (cPLA2, PLA2G4A) catalyzes the release of arachidonic acid for prostaglandin synthesis by cyclooxygenase 1 (PTGS1) and cyclooxygenase 2 (PTGS2). Mice with Pla2g4a deficiency have parturition delay and other reproductive deficits, including deferred onset of implantation, crowding of implantation sites, and small litters. In this study, we examined the contribution of PLA2G4A to parturition in mice. Pla2g4a mRNA and protein expression were discretely localized in the term and preterm uterine luminal epithelium and colocalized with Ptgs1, but not Ptgs2, expression. The levels of PGE2, PGF2alpha, 6-keto-PGF1alpha, and TxB2 were significantly decreased in Pla2g4a-null uterine tissues, similar to Ptgs1-null uteri, consistent with predominance of PLA2G4A-PTGS1-mediated prostaglandin synthesis in preparation for murine parturition. Litter size was strongly associated with the timing of parturition in Pla2g4a-null mice but could not fully account for the parturition delay. Pla2g4a-null females that received PGE2 + carbaprostacyclin at the time of implantation delivered earlier (20.5 +/- 0.2 days vs. 21.6 +/- 0.2 days, P < 0.01), although litter size was not improved (4.6 vs. 4.4 pups per litter, P = 0.6). After correction for small litter size, multivariate analysis indicated that Pla2g4a-null mice given prostaglandin treatment to improve implantation timing had gestational length that was similar to wild-type and Pla2g4a heterozygous mice. These results indicate that, despite specific Pla2g4a expression and function in term gestation uteri, the delayed parturition phenotype in Pla2g4a-null mice is primarily due to deferral of implantation. The role of PLA2G4A in timely parturition appears to be critically related to its actions in early pregnancy.