An atlas of epithelial cell states and plasticity in lung adenocarcinoma.

Understanding the cellular processes that underlie early lung adenocarcinoma (LUAD) development is needed to devise intervention strategies1. Here we studied 246,102 single epithelial cells from 16 early-stage LUADs and 47 matched normal lung samples. Epithelial cells comprised diverse normal and cancer cell states, and diversity among cancer cells was strongly linked to LUAD-specific oncogenic drivers. KRAS mutant cancer cells showed distinct transcriptional features, reduced differentiation and low levels of aneuploidy. Non-malignant areas surrounding human LUAD samples were enriched with alveolar intermediate cells that displayed elevated KRT8 expression (termed KRT8+ alveolar intermediate cells (KACs) here), reduced differentiation, increased plasticity and driver KRAS mutations. Expression profiles of KACs were enriched in lung precancer cells and in LUAD cells and signified poor survival. In mice exposed to tobacco carcinogen, KACs emerged before lung tumours and persisted for months after cessation of carcinogen exposure. Moreover, they acquired Kras mutations and conveyed sensitivity to targeted KRAS inhibition in KAC-enriched organoids derived from alveolar type 2 (AT2) cells. Last, lineage-labelling of AT2 cells or KRT8+ cells following carcinogen exposure showed that KACs are possible intermediates in AT2-to-tumour cell transformation. This study provides new insights into epithelial cell states at the root of LUAD development, and such states could harbour potential targets for prevention or intervention.

Refractive outcomes using Barrett formulas and patient characteristics of cataract surgery patients with and without prior LASIK/PRK.

PURPOSE: The goal of this study is to describe characteristics of cataract surgery patients who previously underwent laser in situ keratomileusis/photorefractive keratectomy (LASIK/PRK) in comparison to non-LASIK/PRK cataract surgery patients including psychiatric comorbidities, as well as describe refractive prediction error after cataract surgery while accounting for axial length (AL) using the Barrett True-K and Barrett Universal II formulas. METHODS: This was a retrospective study of patients from the University of Colorado Cataract Outcomes Registry. The primary outcomes were refraction prediction error (RPE), mean absolute RPE, and median absolute RPE. Outcomes were stratified by five axial length groups. Univariate and multivariate models for RPE were stratified by the AL group. RESULTS: Two hundred eighty-one eyes with prior LASIK/PRK and 3101 eyes without are included in the study. Patients with prior LASIK/PRK were significantly younger: 67.0 vs 69.9 years, p < 0.0001. The LASIK/PRK group had significantly better mean pre-operative BCVA in comparison to the non-LASIK group, logMAR 0.204 vs logMAR 0.288, p = 0.003. The LASIK/PRK group had significantly lower rates of cardiovascular disease (18.5% vs 29.3%, p < 0.001), hypertension (49.1% vs 59.3%, p < 0.012), and type 2 diabetes (10.7% vs 26.0%, p < 0.001), and no significant difference in psychiatric disease. The absolute RPE was higher for the LASIK group for all ALs, but only significantly higher for eyes with AL less than 25 mm. CONCLUSION: Patient eyes with prior LASIK/PRK surgery undergoing cataract surgery were significantly younger, had significantly less comorbidities, and a significantly better pre-operative BCVA. Using the Barrett formulas, absolute prediction error for eyes with longer ALs was not significantly worse for LASIK/PRK eyes than those without and the difference was smaller for eyes with longer AL.

Molecular networks in atopic mothers impact the risk of infant atopy.

BACKGROUND: The prevalence of atopic diseases has increased with atopic dermatitis (AD) as the earliest manifestation. We assessed if molecular risk factors in atopic mothers influence their infants' susceptibility to an atopic disease. METHODS: Pregnant women and their infants with (n = 174, high-risk) or without (n = 126, low-risk) parental atopy were enrolled in a prospective birth cohort. Global differentially methylated regions (DMRs) were determined in atopic (n = 92) and non-atopic (n = 82) mothers. Principal component analysis was used to predict atopy risk in children dependent on maternal atopy. Genome-wide transcriptomic analyses were performed in paired atopic (n = 20) and non-atopic (n = 15) mothers and cord blood. Integrative genomic analyses were conducted to define methylation-gene expression relationships. RESULTS: Atopic dermatitis was more prevalent in high-risk compared to low-risk children by age 2. Differential methylation analyses identified 165 DMRs distinguishing atopic from non-atopic mothers. Inclusion of DMRs in addition to maternal atopy significantly increased the odds ratio to develop AD in children from 2.56 to 4.26. In atopic compared to non-atopic mothers, 139 differentially expressed genes (DEGs) were identified significantly enriched of genes within the interferon signaling pathway. Expression quantitative trait methylation analyses dependent on maternal atopy identified 29 DEGs controlled by 136 trans-acting methylation marks, some located near transcription factors. Differential expression for the same nine genes, including MX1 and IFI6 within the interferon pathway, was identified in atopic and non-atopic mothers and high-risk and low-risk children. CONCLUSION: These data suggest that in utero epigenetic and transcriptomic mechanisms predominantly involving the interferon pathway may impact and predict the development of infant atopy.

A Randomized Prospective Trial Comparing Repository Corticotropin Injection and Intravenous Methylprednisolone for Neuroprotection in Acute Optic Neuritis.

BACKGROUND: Repository corticotrophin injection (RCI, Acthar Gel) and intravenous methylprednisolone (IVMP) improve the rate but not the extent of visual recovery following acute optic neuritis. RCI has adrenal-stimulating and melanocortin receptor-stimulating properties that may endow it with unique anti-inflammatory properties relative to IVMP. METHODS: Individuals with acute optic neuritis of less than 2 weeks duration were prospectively enrolled and randomized 1:1 to receive either RCI or IVMP. Peripapillary retinal nerve fiber layer (pRNFL) and ganglion cell plus inner plexiform layer thickness (GC + IPL) were serially evaluated by OCT. In addition, patient-reported outcomes (PROs) for changes in fatigue, mood, visual function, depression, and quality of life (QOL) were measured, and high and low contrast visual acuity were recorded. RESULTS: Thirty-seven subjects were enrolled (19 RCI; 18 IVMP); the average time from symptom to treatment was 8.8 days. At 6 months, there was no difference in the primary outcome: loss of average pRNFL thickness in the affected eye (RCI vs IVMP: -13.1 vs -11.7 µm, P = 0.88) 6 months after randomization. Additional outcomes also showed no difference between treatment groups: 6-month attenuation of GC + IPL thickness (RCI vs IVMP: -13.8 vs -12.0 µm, P = 0.58) and frequency of pRNFL swelling at 1 month (RCI vs IVMP: 63% vs 72%, P = 0.73) and 3 months (RCI vs IVMP: 26% vs 31%, P = 0.99). Both treatments resulted in improvement in visual function and PROs. CONCLUSIONS: Treatment of acute optic neuritis with RCI or IVMP produced no clinically meaningful differences in optic nerve structure or visual function.

Intermediary Role of Lung Alveolar Type 1 Cells in Epithelial Repair upon Sendai Virus Infection.

The lung epithelium forms the first barrier against respiratory pathogens and noxious chemicals; however, little is known about how more than 90% of this barrier, made of AT1 (alveolar type 1) cells, responds to injury. Using the Sendai virus to model natural infection in mice, we find evidence that AT1 cells have an intermediary role by persisting in areas depleted of AT2 cells, upregulating IFN responsive genes, and receding from invading airway cells. Sendai virus infection mobilizes airway cells to form alveolar SOX2+ (Sry-box 2+) clusters without differentiating into AT1 or AT2 cells. Large AT2 cell-depleted areas remain covered by AT1 cells, which we name "AT2-less regions", and are replaced by SOX2+ clusters spreading both basally and luminally. AT2 cell proliferation and differentiation are largely confined to topologically distal regions and form de novo alveolar surface, with limited contribution to in situ repairs of AT2-less regions. Time-course single-cell RNA sequencing profiling and RNAscope validation suggest enhanced immune responses and altered growth signals in AT1 cells. Our comprehensive spatiotemporal and genomewide study highlights the hitherto unappreciated role of AT1 cells in lung injury-repair.

Mental health and visual acuity in patients with age-related macular degeneration.

BACKGROUND: Visual acuity (VA) loss has been associated with depression in patients with age-related macular degeneration (AMD). However, previous studies did not incorporate subgroups of AMD when correlating VA and mental health. The goal of this study was to describe the relationship between VA and mental health questions in patients with different classifications of AMD, and to identify associations of mental health subscale scores. METHODS: AMD patients classified by multi-modal imaging were recruited into an AMD registry. Habitual VA was obtained by ophthalmic technicians using the Snellen VA at distance. At enrollment, patients completed the NEI-VFQ-25, which includes 25 questions regarding the patient's visual functionality. Median with interquartile-range (IQR) scores on the mental health subscale of the VFQ were calculated by AMD classification and VA groups. Univariate and multivariable general linear models were used to estimate associations between mental health scores and variables of interest. RESULTS: Eight hundred seventy-five patients were included in the study. Patients with bilateral geographic atrophy (GA) or bilateral GA and neovascular (NV) AMD scored lowest on the mental health subscales with a median (IQR) of 58.2 (38-88) and 59.3 (38-88). When stratified by VA, patients with a habitual VA of 20/200 or worse scored the lowest on mental health subscales scores: median of 43.8 (IQR: 31-62). Patients with a VA of 20/20 scored the highest: 87.5 (IQR: 81-94). Habitual VA of the better- and worse-seeing eye and AMD classification were significantly associated with mental health subscale scores (all p < 0.0001 in both the univariate and multivariable analysis, except the VA of the worse-seeing eye in multivariable model p = 0.027). Patients enrolled during the COVID pandemic had mental health scores that were 2.7 points lower than prior to the pandemic, but this difference was not significant in univariate (p = 0.300) or multivariable analysis (p = 0.202). CONCLUSION: There is a significant association between mental health questionnaire scores and AMD classification, as well as VA in both the better and worse-seeing eyes in patients with AMD. It is important for clinicians to recognize feelings of worry/ frustration in these patients, so they can be appropriately referred, screened, and treated for mental health problems.

An Exploratory Study to Investigate the Utility of Clinical Screening for Neurodegenerative Disease in Age-Related Eye Disease Research.

BACKGROUND: Unrecognized neurodegenerative diseases (NDD) in age-related eye disease research studies have the potential to confound vision-specific quality of life and retinal optical coherence tomography (OCT) outcome measures. The aim of this exploratory study was to investigate relationships between NDD screening tools and visual outcome measures in a small cohort of controls from the Colorado Age-Related Macular Degeneration Registry (CO-AMD), to consider the utility of future studies. METHODS: Twenty-nine controls from the CO-AMD were screened using the Montreal Cognitive Assessment (MoCA), a Colorado Parkinsonian Checklist, and the Lewy Body Composite Risk Score. Univariate and multivariable linear regression modeling was used to assess associations between screening tools and the National Eye Institute Visual Function Questionnaire-25 (VFQ-25) and macular OCT outcome measures, and t tests were used to evaluate outcome measure differences between those with normal vs abnormal MoCA scores. RESULTS: One patient withdrew. The average age was 72.8 years, and 68% were female patients. Ten participants (36%) had abnormal MoCA scores, and their VFQ-25 scores were only 1 point less and not statistically different than those with normal MoCA scores. Macular OCT volumes and thicknesses for retinal nerve fiber layer (RNFL) and retinal ganglion cell layer were consistently and moderately lower for those with abnormal MoCA scores, and a positive association between MoCA and macular RNFL volume was observed, although differences and regression were not significant. Parkinson screening tests were abnormal for only 4 participants and were not associated with OCT or VFQ-25 measures by regression modeling. CONCLUSIONS: Given the degree and direction of observed differences, further investigation is warranted regarding the relationship between cognitive screening tools and macular OCT measures in age-related eye disease research, but future investigations regarding the relationship between NDD screening tools and VFQ-25 seem unwarranted.

COLORADO AGE-RELATED MACULAR DEGENERATION REGISTRY: Design and Clinical Risk Factors of the Cohort.

PURPOSE: To study new and existing risk factors related to age-related macular degeneration (AMD) phenotypes in a Colorado cohort. METHODS: Age-related macular degeneration was categorized into early, intermediate, or advanced forms. Controls (n = 180) were patients with cataract and no AMD. Demographic and clinical data were gathered by patient interview and verified by chart review. Image data were reviewed by vitreoretinal specialists. Statistical analysis included univariable and multivariate logistic regression analysis (P < 0.05). RESULTS: Among the 456 patients with AMD, 157 (34.4%), 80 (17.6%), and 219 (48.0%) had the early/intermediate, geographic atrophy, and neovascular forms of the disease, respectively. Adjusted for age, African-American race was associated with a reduced risk of early/intermediate (adjusted odds ratio [AOR] = 0.08, confidence interval [CI] = 0.01-0.67) and neovascular AMD (AOR = 0.15, CI = 0.03-0.72). A family history of AMD was a risk factor for early/intermediate (AOR = 4.08, CI = 2.30-7.25), geographic atrophy (AOR = 8.62, CI = 3.77-19.7), and neovascular AMD (AOR = 3.76, CI = 2.16-6.56). A history of asthma was related to the early/intermediate form of AMD (AOR = 2.34, CI = 1.22-4.46). CONCLUSION: Studying AMD in specific populations may reveal novel risk factors such as our finding of a relationship between asthma history and AMD.

Shorter Time to Pregnancy With Increasing Preconception Carotene Concentrations Among Women With 1-2 Previous Pregnancy Losses.

Although maternal nutrition may affect fecundity, associations between preconception micronutrient levels and time to pregnancy (TTP) have not been examined. We assessed the relationship between preconception fat-soluble micronutrient concentrations and TTP among women with 1-2 prior pregnancy losses. This was a prospective cohort study of 1,228 women set within the Effects of Aspirin in Gestation and Reproduction (EAGeR) Trial (United States, 2007-2011), which assessed the association of preconception-initiated daily low-dose aspirin with reproductive outcomes. We measured preconception levels of zeaxanthin, cryptoxanthin, lycopene, α- and ß-carotene, and α- and γ-tocopherol in serum. We used discrete Cox regression models, accounting for left-truncation and right-censoring, to calculate fecundability odds ratios and 95% confidence intervals. The models adjusted for age, body mass index, race, smoking, alcohol, physical activity, income, vitamin use, cholesterol, treatment arm, and study site. Serum α-carotene levels (per log unit (µg/dL) increase, fecundability odds ratio (FOR) = 1.17, 95% confidence interval (CI): 1.00, 1.36) and serum α-carotene concentrations at or above the US average (2.92 µg/dL) versus below the average (FOR = 1.21, 95% CI: 1.02, 1.44) were associated with shorter TTP. Compared with levels below the US average (187 µg/dL), γ-tocopherol concentrations at or above the average were associated with longer TTP (FOR = 0.83, 95% CI: 0.69, 1.00). The potential for these nutrients to influence fecundability deserves further exploration.

Complement activation predicts adverse pregnancy outcome in patients with systemic lupus erythematosus and/or antiphospholipid antibodies.

OBJECTIVE: Studies in mouse models implicate complement activation as a causative factor in adverse pregnancy outcomes (APOs). We investigated whether activation of complement early in pregnancy predicts APOs in women with systemic lupus erythematosus (SLE) and/or antiphospholipid (aPL) antibodies. METHODS: The PROMISSE Study enrolled pregnant women with SLE and/or aPL antibodies (n=487) and pregnant healthy controls (n=204) at <12 weeks gestation and evaluated them monthly. APOs were: fetal/neonatal death, preterm delivery <36 weeks because of placental insufficiency or preeclampsia and/or growth restriction <5th percentile. Complement activation products were measured on serial blood samples obtained at each monthly visit. RESULTS: APO occurred in 20.5% of SLE and/or aPL pregnancies. As early as 12-15 weeks, levels of Bb and sC5b-9 were significantly higher in patients with APOs and remained elevated through 31 weeks compared with those with normal outcomes. Moreover, Bb and sC5b-9 were significantly higher in patients with SLE and/or aPL without APOs compared with healthy controls. In logistic regression analyses, Bb and sC5b-9 at 12-15 weeks remained significantly associated with APO (ORadj=1.41 per SD increase; 95% CI 1.06 to 1.89; P=0.019 and ORadj=1.37 per SD increase; 95% CI 1.05 to 1.80; P=0.022, respectively) after controlling for demographic and clinical risk factors for APOs in PROMISSE. When analyses were restricted to patients with aPL (n=161), associations between Bb at 12-15 weeks and APOs became stronger (ORadj=2.01 per SD increase; 95% CI 1.16 to 3.49; P=0.013). CONCLUSION: In pregnant patients with SLE and/or aPL, increased Bb and sC5b-9 detectable early in pregnancy are strongly predictive of APOs and support activation of complement, particularly the alternative pathway, as a contributor to APOs.

Preconception maternal lipoprotein levels in relation to fecundability.

STUDY QUESTION: Are maternal preconception lipid levels associated with fecundability? SUMMARY ANSWER: Fecundability was reduced for all abnormal female lipid levels including total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and total triglyceride levels. WHAT IS KNOWN ALREADY: Subfecundity affects 7-15% of the population and lipid disorders are hypothesized to play a role since cholesterol acts as a substrate for the synthesis of steroid hormones. Evidence illustrating this relationship at the mechanistic level is mounting but few studies in humans have explored the role of preconception lipids in fecundity. STUDY DESIGN, SIZE, DURATION: A secondary analysis of the Effects of Aspirin in Gestation and Reproduction (EAGeR) trial (2007-2011), a block-randomized, double-blind, placebo-controlled trial. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 1228 women, with 1-2 prior pregnancy losses and without a diagnosis of infertility, attempting pregnancy for up to six menstrual cycles were recruited from clinical sites in Utah, New York, PA and Colorado. Time to pregnancy was the number of menstrual cycles to pregnancy as determined by positive hCG test or ultrasound. Individual preconception lipoproteins were measured at baseline, prior to treatment randomization and dichotomized based on clinically accepted cut-points as total cholesterol ≥200 mg/dl, LDL-C ≥130 mg/dl, HDL-C <50 mg/dl and triglycerides ≥150 mg/dl. MAIN RESULTS AND THE ROLE OF CHANCE: There were 148 (12.3%) women with elevated total cholesterol, 94 (7.9%) with elevated LDL-C, 280 (23.2%) with elevated triglycerides and 606 (50.7%) with low HDL-C. The fecundability odds ratio (FOR) was reduced for all abnormal lipids before and after confounder adjustment, indicating reduced fecundability. Total cholesterol ≥200 mg/dl was associated with 24% (FOR: 0.76, 95% CI: 0.59, 0.97) and 29% (FOR: 0.71, 95% CI: 0.55, 0.93) reduced fecundability for hCG-detected and ultrasound-confirmed pregnancy, respectively, compared with total cholesterol <200 mg/dl. There was a 19-36% decrease in the probability of conception per cycle for women with abnormal lipoprotein levels, though additional adjustment for central adiposity and BMI attenuated observed associations. LIMITATIONS, REASONS FOR CAUTION: Although the FOR is a measure of couple fecundability, we had only measures of female lipid levels and can therefore not confirm the findings from a previous study indicating the independent role of male lipids in fecundity. The attenuated estimates and decreased precision after adjustment for central adiposity and obesity indicate the complexity of potential causal lipid pathways, suggesting other factors related to obesity besides dyslipidemia likely contribute to reduced fecundability. WIDER IMPLICATIONS OF THE FINDINGS: Our results are consistent with one other study relating preconception lipid concentrations to fecundity and expand these findings by adding critically important information about individual lipoproteins. As lipid levels are modifiable they may offer an inexpensive target to improve female fecundability. STUDY FUNDING AND COMPETING INTEREST(S): This study was funded by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development. The authors have declared that no conflicts of interest exist. TRIAL REGISTRATION NUMBER: #NCT00467363.

The relationship of the subtypes of preterm birth with retinopathy of prematurity.

BACKGROUND: Retinopathy of prematurity is an adverse outcome of preterm birth and is a leading cause of childhood blindness. The relationship between the subtypes of preterm birth with retinopathy of prematurity is understudied. OBJECTIVE: To investigate whether there is a difference in the incidence of type 1 or type 2 retinopathy of prematurity in infants with preterm birth resulting from spontaneous preterm labor, a medical indication of preterm birth, or preterm premature rupture of the membranes. STUDY DESIGN: A retrospective cohort study was conducted of 827 infants screened for retinopathy of prematurity who were delivered at a single tertiary care center in Colorado. All infants fulfilled the American Academy of Pediatrics 2013 screening criteria for retinopathy of prematurity defined as "infants with a birth weight of ≤1500 g or gestational age of 30 weeks or less (as defined by the attending neonatologist) and selected infants with a birth weight between 1500 and 2000 g or gestational age of >30 weeks with an unstable clinical course, including those requiring cardiorespiratory support and who are believed by their attending pediatrician or neonatologist to be at high risk for retinopathy of prematurity." Two independent reviewers masked to retinopathy of prematurity outcomes determined whether preterm birth resulted from spontaneous preterm labor, medical indication of preterm birth, or preterm premature rupture of the membranes. Discrepancies were resolved by a third reviewer. Data were analyzed with univariate and multivariable logistic regression. RESULTS: In our cohort, the frequency of preterm birth resulting from spontaneous preterm labor, medical indication of preterm birth, or preterm premature rupture of the membranes was 34%, 40%, and 26%, respectively. The mean gestational age (weeks, days) ± SD (range) in the cohort and across the preterm birth subtypes was as follows: entire cohort, 28 weeks, 6 days ± 2 weeks, 3 days (23 weeks, 3 days - 36 weeks, 4 days); spontaneous preterm labor, 28 weeks 1 day ± 2 weeks, 3 days (23 weeks, 3 days - 33 weeks, 4 days); medical indication of preterm birth, 29 weeks, 1 day ± 2 weeks, 2 days (24-36 weeks, 4 days); preterm premature rupture of the membranes, 28 weeks, 4 days ± 2 weeks, 1 day (24-33 weeks, 1 day). Among infants with type 1, type 2, or no retinopathy of prematurity, the incidence of type 1 or type 2 retinopathy of prematurity in births from spontaneous preterm labor, medical indication of preterm birth, and preterm premature rupture of the membranes was 37 of 218 (17%), 27 of 272 (10%), and 10 of 164 (6%), respectively. Adjusted for gestational age, birth weight, and multiparity and compared with the preterm premature rupture of the membranes group, the odds ratios of spontaneous preterm labor and medical indication of preterm birth for type 1 or type 2 retinopathy of prematurity were 6.1 (95% confidence interval, 1.8 to 20, P = .003) and 5.5 (95% confidence interval, 1.4 to 21, P = .01), respectively. Among neonates born after preterm premature rupture of the membranes, the probability of developing type 1 or type 2 retinopathy of prematurity was greatest in infants with rupture of membrane duration of up to 24 hours. After 24 hours, the probability of developing type 1 or type 2 retinopathy of prematurity declined. The odds of developing type 1 or type 2 retinopathy of prematurity was 9.0 (95% confidence interval 2.3 to 34, P = .002) in infants who had preterm premature rupture of the membranes ≤ 24 hours compared with infants who had preterm premature rupture of the membranes > 24 hours. CONCLUSION: Type 1 or type 2 retinopathy of prematurity are adverse ocular outcomes linked with not only lower gestational age and birth weight at delivery but also with events in the intrauterine environment that trigger a preterm birth. The reduced incidence of type 1 or type 2 retinopathy of prematurity in the preterm premature rupture of the membranes group compared with other causes of preterm birth may be related to the perinatal therapies associated with preterm premature rupture of the membranes (such as corticosteroids, antibiotics, maternal-fetal surveillance), which may have an inhibitory effect on the development of retinopathy of prematurity. We suggest that the physiologic events that predispose infants to type 1 or type 2 retinopathy of prematurity begin before delivery.

Expanded findings from a randomized controlled trial of preconception low-dose aspirin and pregnancy loss.

STUDY QUESTION: What is the association between daily preconception-initiated low-dose aspirin (LDA) treatment and very early pregnancy losses or euploid (chromosomally normal) losses among women with one to two prior losses? SUMMARY ANSWER: Daily LDA initiated preconception was not associated with the rate or type of pregnancy loss among women with a history of one to two prior pregnancy losses. WHAT IS KNOWN ALREADY: LDA is often used to treat recurrent pregnancy loss with reductions in pregnancy loss generally only observed among women with antiphospholipid antibodies, and null associations observed among women without antiphospholipid antibodies. We previously evaluated the association between LDA and pregnancy loss overall among women with one to two prior losses in the Effects of Aspirin in Gestation and Reproduction (EAGeR) trial and found no association, though did not distinguish between potential effects at different stages of pregnancy loss, including implantation failure, or between euploid and aneuploid losses. STUDY DESIGN, SIZE, DURATION: The EAGeR trial was a multi-site prospective block-randomized double-blind placebo-controlled trial. In total, 1228 women were randomized to daily LDA (81 mg/day) plus folic acid (400 mcg/day), or placebo plus folic acid. Participants were assigned study drug for less than or equal to six menstrual cycles or if they conceived, throughout pregnancy with study drug discontinued at 36 weeks gestation. This analysis includes additional outcome information obtained from chart abstractions after the completion of the trial, as well as testing of stored urine for measurement of hCG and detection of very early pregnancy losses, and karyotyping of the products of conception for assessment of aneuploidy of the losses. PARTICIPANTS, SETTING, METHODS: Women aged 18-40 with a history of one to two prior losses and actively trying to conceive were randomized (n = 615 LDA and n = 613 placebo) at four clinical centers in the USA (2007-2011). Log-binomial regression was used to estimate risk ratios under the intent-to-treat approach. MAIN RESULTS AND THE ROLE OF CHANCE: Daily LDA initiated preconception was not associated with clinically recognized pregnancy losses or implantation failures among women with proved fecundity and a history of one to two prior losses. Specifically, 1088 (88.6%) women completed the trial with 797 having an hCG detected pregnancy (64.9%). Overall there were 133 clinical losses (12.7% LDA versus 11.8% placebo, P = 0.71) and 55 implantation failures (5.2% LDA versus 4.9% placebo, P = 0.89). No differences were found in rate of euploid losses (RR 1.11, 95% confidence interval: 0.99, 1.26). LIMITATIONS, REASONS FOR CAUTION: Generalizability of these findings is limited to women with a history of one to two prior losses, and may further be limited to women of white race with higher socioeconomic status as given the rigors of the study protocol participants tended to be white and have higher incomes and more education. We were also missing karyotype information on approximately one-third of the clinically recognized pregnancy losses, which may limit our power to detect effects on euploid losses, though detailed sensitivity analysis showed similar results. WIDER IMPLICATIONS OF THE FINDINGS: Our data do not support the general use of LDA to decrease pregnancy loss and further demonstrate no increased risk of loss for women on LDA treatment. STUDY FUNDING/COMPETING INTERESTS: This research was supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland (Contract Nos. HHSN267200603423, HHSN267200603424, HHSN267200603426). The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: The trial was registered at ClinicalTrials.gov #NCT00467363. TRIAL REGISTRATION DATE: 27 April 2007. DATE OF FIRST PATIENT'S ENROLLMENT: 15 June 2007.

The relationship of circulating proteins in early pregnancy with preterm birth.

BACKGROUND: Preterm birth (PTB) (< 37 completed weeks' gestation) is a pathological outcome of pregnancy and a major global health problem. Babies born preterm have an elevated risk for long-term adverse medical and neurodevelopmental sequelae. Substantial evidence implicates intrauterine infection and/or inflammation in PTB. However, these are often relatively late findings in the process, when PTB is inevitable. Identification of earlier markers of PTB may make successful intervention possible. Although select proteins, notably those related to the inflammatory pathways, have been associated with PTB, there has been a lack of research into the role of other protein pathways in the development of PTB. The purpose of this study was to investigate, using a previously described biomarker discovery approach, a subset of circulating proteins and their association with PTB focusing on samples from early pregnancy. OBJECTIVES: The objectives of the study were as follows: (1) to perform a large-scale biomarker discovery, utilizing an innovative platform to identify proteins associated with preterm birth in plasma taken between 10 and 15 weeks' gestation and, (2) to determine which protein pathways are most strongly associated with preterm birth. To address these aims, we measured 1129 proteins in a plasma sample from early pregnancy using a multiplexed aptamer-based proteomic technology developed in Colorado by SomaLogic. STUDY DESIGN: Using a nested case-control approach, we measured proteins at a single time point in early pregnancy in 41 women who subsequently delivered preterm and 88 women who had term uncomplicated deliveries. We measured 1129 proteins using a multiplexed aptamer-based proteomic technology developed by SomaLogic. Logistic regressions and random forests were used to compare protein levels. RESULTS: The complement factors B and H and the coagulation factors IX and IX ab were the highest-ranking proteins distinguishing cases of preterm birth from term controls. The top 3 pathways associated with preterm birth were the complement cascade, the immune system, and the clotting cascade. CONCLUSION: Using a discovery approach, these data provide further confirmation that there is an association of immune- and coagulation-related events in early pregnancy with preterm birth. Thus, plasma protein profiles at 10-15 weeks of gestation are related to the development of preterm birth later in pregnancy.

Preconception low-dose aspirin and pregnancy outcomes: results from the EAGeR randomised trial.

BACKGROUND: Preconception-initiated low-dose aspirin might positively affect pregnancy outcomes, but this possibility has not been adequately assessed. Our aim was to investigate whether low-dose aspirin improved livebirth rates in women with one to two previous pregnancy losses. METHODS: In this multicentre, block-randomised, double-blind, placebo-controlled trial, women aged 18-40 years who were attempting to become pregnant were recruited from four medical centres in the USA. Participants were stratified by eligibility criteria--the original stratum was restricted to women with one loss at less than 20 weeks' gestation during the previous year, whereas the expanded stratum included women with one to two previous losses, with no restrictions on gestational age or time of loss. Women were block-randomised by centre and eligibility stratum in a 1:1 ratio. Preconception-initiated daily low-dose aspirin (81 mg per day) plus folic acid was compared with placebo plus folic acid for up to six menstrual cycles; for women who conceived, study treatment continued until 36 weeks' gestation. Participants, trial staff, and investigators were masked to the assigned treatment. The primary outcome was livebirth rate, which was analysed by intention to treat. The trial is registered with ClinicalTrials.gov, number NCT00467363. FINDINGS: Overall, 1228 women were recruited and randomly assigned between June 15, 2007, and July 15, 2011, 1078 of whom completed the trial and were included in the analysis (535 in the low-dose aspirin group and 543 in the placebo group). 309 (58%) women in the low-dose aspirin group had livebirths, compared with 286 (53%) in the placebo group (p=0·0984; absolute difference in livebirth rate 5·09% [95% CI -0·84 to 11·02]). Pregnancy loss occurred in 68 (13%) women in the low-dose aspirin group, compared with 65 (12%) women in the placebo group (p=0·7812). In the original stratum, 151 (62%) of 242 women in the low-dose aspirin group had livebirths, compared with 133 (53%) of 250 in the placebo group (p=0·0446; absolute difference in livebirth rate 9·20% [0·51 to 17·89]). In the expanded stratum, 158 (54%) of 293 women in the low-dose aspirin group and 153 (52%) of 293 in the placebo group had livebirths (p=0·7406; absolute difference in livebirth rate 1·71% [-6·37 to 9·79]). Major adverse events were similar between treatment groups. Low-dose aspirin was associated with increased vaginal bleeding, but this adverse event was not associated with pregnancy loss. INTERPRETATION: Preconception-initiated low-dose aspirin was not significantly associated with livebirth or pregnancy loss in women with one to two previous losses. However, higher livebirth rates were seen in women with a single documented loss at less than 20 weeks' gestation during the previous year. Low-dose aspirin is not recommended for the prevention of pregnancy loss. FUNDING: Eunice Kennedy Shriver National Institute of Child Health and Human Development (US National Institutes of Health).

The effect of a very short interpregnancy interval and pregnancy outcomes following a previous pregnancy loss.

OBJECTIVE: We sought to assess the relationship between a short interpregnancy interval (IPI) following a pregnancy loss and subsequent live birth and pregnancy outcomes. STUDY DESIGN: A secondary analysis of women enrolled in the Effects of Aspirin in Gestation and Reproduction trial with a human chorionic gonadotropin-positive pregnancy test and whose last reproductive outcome was a loss were included in this analysis (n = 677). IPI was defined as the time between last pregnancy loss and last menstrual period of the current pregnancy and categorized by 3-month intervals. Pregnancy outcomes include live birth, pregnancy loss, and any pregnancy complications. These were compared between IPI groups using multivariate relative risk estimation by Poisson regression. RESULTS: Demographic characteristics were similar between IPI groups. The mean gestational age of prior pregnancy loss was 8.6 ± 2.8 weeks. The overall live birth rate was 76.5%, with similar live birth rates between those with IPI ≤3 months as compared to IPI >3 months (adjusted relative risk [aRR], 1.07; 95% confidence interval [CI], 0.98-1.16). Rates were also similar for periimplantation loss (aRR, 0.95; 95% CI, 0.51-1.80), clinically confirmed loss (aRR, 0.75; 95% CI, 0.51-1.10), and any pregnancy complication (aRR, 0.88; 95% CI, 0.71-1.09) for those with IPI ≤3 months as compared to IPI >3 months. CONCLUSION: Live birth rates and adverse pregnancy outcomes, including pregnancy loss, were not associated with a very short IPI after a prior pregnancy loss. The traditional recommendation to wait at least 3 months after a pregnancy loss before attempting a new pregnancy may not be warranted.

Recruitment for longitudinal, randomised pregnancy trials initiated preconception: lessons from the effects of aspirin in gestation and reproduction trial.

BACKGROUND: Recruitment into large, preconception randomised clinical trials (RCT) is challenging. We describe clinic and community-based preconception recruitment strategies for the Effects of Aspirin in Gestation and Reproduction (EAGeR) trial and highlight approaches that were and were not successful. This longitudinal RCT was conducted at four major sites in the US. Eligible women had one to two prior pregnancy losses and were actively trying to become pregnant. METHODS: Provider/clinic and community-based outreach strategies were utilised, and the recruitment rate and costs of methods were assessed. RESULTS: A screening questionnaire was completed by 5485 women; 42.4% (n = 2323) screened were initially eligible, of whom 50.7% (n = 1228) were randomised. Provider/clinic-based recruitment yielded the highest number eligible of those screened (30.1%) and also the most randomised participants overall (40.3%). The next highest yield came from direct mail and brochures/flyers at 13.1% and 12.5% of women randomised, respectively. However, direct mailings cost $720 per participant randomised. Other than word of mouth, provider/clinic-based recruitment was the most cost effective method, costing an average of $60 per randomised participant. Web-based recruitment yielded 4.7% of participants at a cost of $278 per randomised participant. CONCLUSIONS: Provider and clinic-based recruitment was the most effective and cost-efficient method of recruitment in a preconception intervention study of reproduction among women.