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Additional file 1: Figure S1. Study activities overview: overview of all study activities. Figure S2. Sample processing in PRIMA human milk cohort: A) Human milk samples are centrifuged for 10 min at 600 g in order to remove the cream (layer on top, indicated in yellow) and cells (collected in pellet on the bottom, indicated in dark grey). B) Milk supernatant is collected by pipetting through cream layer without disturbing the cell pellet and subsequently transferred into a new tube. C) Collected 600 g supernatant is again centrifuged for another 10 min at 600 g. D) Milk supernatant is collected by pipetting through the residual cream and transferred to and aliquoted into clean cryotubes, and stored at -80 ��C until further analysis. E) The cream layer is removed without disturbing the cellular pellet and transferred into cryotubes stored at -80 ��C for further analysis. F) 20 mL PBS is added to rinse and cells are spun at 600 g for 10 min, after which the supernatant is discarded and cells are kept on ice until further processing for analysis G). Milk EVs are processed further according to the Zonneveld protocol at 3000 g.

Additional file 2. PRIMA research protocol METC Utrecht version 4A: this file contains the protocol including the novel additions for collection of dietary information from mother.

Results from pragmatic trials should reflect the comparative treatment effects encountered in patients in real-life clinical practice to guide treatment decisions. Therefore pragmatic trials should focus on outcomes that are relevant to patients, clinical practice, and treatment choices. This 6(th) paper in the series (see box) discusses different types of outcomes and their suitability for pragmatic trials, design choices for measuring these outcomes and their implications and challenges. Measuring outcomes in pragmatic trials should not interfere with real-world clinical practice to ensure generalizability of trial results and routinely collected outcomes should be prioritized. Typical outcomes include mortality, morbidity, functional status, wellbeing and resource use. Surrogate endpoints are typically avoided as primary outcome. It is important to measure outcomes over a relevant time horizon and to obtain valid and precise results. As pragmatic trials are often open-label a less subjective outcome can reduce bias. Methods that decrease bias or enhance precision of the results, such as standardization and blinding of outcome assessment, should be considered when a high risk of bias or high variability is expected. The selection of outcomes in pragmatic trials should be relevant for decision making as well as feasible in terms of executing the trial in the context of interest. Therefore this should be discussed with all stakeholders as early as feasible to ensure the relevance of study results for decision making in clinical practice and the ability to perform the study.

This second article in the series on pragmatic trials describes the challenges in selection of sites for pragmatic clinical trials and the impact on validity, precision, and generalizability of the results. The selection of sites is an important factor for the successful execution of a pragmatic trial and impacts the extent to which the results are applicable to future patients in clinical practice. The first step is to define usual care and understand the heterogeneity of sites, patient demographics, disease prevalence and country choice. Next, specific site characteristics are important to consider such as interest in the objectives of the trial, the level of research experience, availability of resources, and the expected number of eligible patients. It can be advisable to support the sites with implementing the trial-related activities and minimize the additional burden that the research imposes on routine clinical practice. Health care providers should be involved in an early phase of protocol development to generate engagement and ensure an appropriate selection of sites with patients who are representative of the future drug users.

OBJECTIVE: To assess the effectiveness of curettage versus expectant management in women with an incomplete evacuation of the uterus after misoprostol treatment for first trimester miscarriage. STUDY DESIGN: We conducted a multicenter cohort study alongside a randomized clinical trial (RCT) between June 2012 until July 2014. 27 Dutch hospitals participated. Women with an incomplete evacuation after misoprostol treatment for first trimester miscarriage who declined to participate in the RCT, received treatment of their preference; curettage (n=65) or expectant management (n=132). A successful outcome was defined as an empty uterus on sonography at six weeks or uneventful clinical follow-up. We furthermore assessed complication rate and (re)intervention rate RESULTS: Of the 197 women who declined to participate in the RCT, 65 preferred curettage and 132 expectant management. A successful outcome was observed in 62/65 women (95%) in the surgical group versus 112/132 women (85%) in the expectant group (RR 1.1, 95% CI 1.03-1.2), with complication rates of 6.2% versus 2.3%, respectively (RR 2.7, 95% CI 0.6-12). CONCLUSION: In women with an incomplete evacuation of the uterus after misoprostol treatment, expectant management is an effective and safe option. This finding could restrain the use of curettage in women that have used misoprostol in the treatment of first trimester miscarriage.

Results from pragmatic trials should reflect the comparative treatment effects encountered in patients in real-life clinical practice to guide treatment decisions. Therefore, pragmatic trials should focus on outcomes that are relevant to patients, clinical practice, and treatment choices. This sixth article in the series (see box) discusses different types of outcomes and their suitability for pragmatic trials, design choices for measuring these outcomes, and their implications and challenges. Measuring outcomes in pragmatic trials should not interfere with real-world clinical practice to ensure generalizability of trial results, and routinely collected outcomes should be prioritized. Typical outcomes include mortality, morbidity, functional status, well-being, and resource use. Surrogate endpoints are typically avoided as primary outcome. It is important to measure outcomes over a relevant time horizon and obtain valid and precise results. As pragmatic trials are often open label, a less subjective outcome can reduce bias. Methods that decrease bias or enhance precision of the results, such as standardization and blinding of outcome assessment, should be considered when a high risk of bias or high variability is expected. The selection of outcomes in pragmatic trials should be relevant for decision making and feasible in terms of executing the trial in the context of interest. Therefore, this should be discussed with all stakeholders as early as feasible to ensure the relevance of study results for decision making in clinical practice and the ability to perform the study.

Objective: Preterm birth is the most common cause of neonatal morbidity and mortality. Around one third of preterm deliveries starts with preterm prelabor rupture of membranes (PPROM). The aim of this trial was to study the effect of prolonged tocolysis with nifedipine versus placebo in women with PPROM on perinatal outcome and prolongation of pregnancy. Study design: The Apostel IV was a nationwide multicenter randomized placebo controlled trial. We included women with PPROM without contractions between 24(+0) and 33(+6) weeks of gestation. Participants were randomly allocated to daily 80 mg nifedipine or placebo, until the start of labor, with a maximum of 18 days. The primary outcome measure was a composite of poor neonatal outcome, including perinatal death, bronchopulmonary dysplasia, periventricular leukomalacia >grade 1, intraventricular hemorrhage >grade 2, necrotizing enterocolitis > stage 1 and culture proven sepsis. Secondary outcomes were gestational age at delivery and prolongation of pregnancy. Analysis was by intention to treat. To detect a reduction of poor neonatal outcome from 30% to 10%,120 women needed to be randomized. Trial registry: NTR 3363. Results: Between October 2012 and December 2014 we randomized 25 women to nifedipine and 25 women to placebo. Due to slow recruitment the study was stopped prematurely. The median gestational age at randomization was 29.9 weeks (IQR 27.7-31.3) in the nifedipine group and 27.0 weeks (IQR 24.7-29.9) in the placebo group. Other baseline characteristics were comparable. The adverse perinatal outcome occurred in 9 neonates (33.3%) in the nifedipine group and 9 neonates (32.1%) in the placebo group (RR 1.04, 95% CI 0.49-2.2). Two perinatal deaths occurred, both in the nifedipine group. Bronchopulmonary dysplasia was seen less frequently in the nifedipine group (0% versus 17.9%; p= 0.03). Prolongation of pregnancy did not differ between the nifedipine and placebo group (median 11 versus 8 days, HR 1.02; 95% CI 0.58-1.79). Conclusion: This randomized trial did not show a beneficial effect of prolonged tocolysis on neonatal outcomes or prolongation of pregnancy in women with PPROM without contractions. However, since results are based on a small sample size, a difference in effectiveness cannot be excluded. Crown Published by

AIM: To describe the vaginal microcirculatory architecture and capillary density parameters using sidestream dark-field imaging (SDFI), and determine feasibility and reliability of this method. METHODS: In nine healthy female volunteers SDFI measurements were performed at two different time points in the luteal phase of the menstrual cycle. Non-invasive tissue micro-angioarchitecture and vaginal capillary density measurements were assessed independently by two observers. Agreement was expressed with mean differences between the measurements of both observers and the limits of agreement. Inter- and intra-observer agreement was quantified with the intra-class correlation coefficient (ICC). RESULTS: Vaginal microcirculatory assessment with the SDFI device was easy in use, painless and well accepted by the participants. Morphologically, the vaginal microcirculation revealed an array of single hairpin-shaped capillary loops distributed homogeneously across an imaged tissue segment. The intra-observer assessment of the capillary density measurements (comparing two measurement time points of one observer) showed good agreement with an ICC ranging from 0.62 to 0.85. The inter-observer assessments of the capillary density measurements (comparing assessments of two observers at one time point) revealed very good agreement, with small differences between observers and an ICC of more than 0.9. CONCLUSIONS: This is the first report on both microcirculatory architecture and quantitative microcirculatory parameters of the vagina with the use of SDFI. Micro-vessels of the vagina show a recognizable pattern in our study population of young, healthy women. SDFI gives a reproducible assessment of the vaginal microcirculation offering the researcher a wide field of applications. Neurourol. Urodynam. 34:723-729, 2015. © 2014 Wiley Periodicals, Inc.