2. Materials and Methods
gastrointestinal symptoms, venous umbilical cord blood sample, WG
INFLAMMATION, BLOOD, RECRUITMENT, SECONDARY, EVENTS
Study design. This was a two-arm double-blind placebo-controlled randomized supplementation feasibility study conducted at an urban Midwest academic medical center. All participants provided written informed consent (IRB #2016-0662) prior to study participation. The study is registered at Research participants. Clinic schedules were reviewed daily to identify potentially eligible women. Initial eligibility was further assessed via electronic health records (EHR). Women identified as potentially eligible were approached in clinic or called to assess interest. Inclusion and exclusion criteria are presented in Treatment groups. Women were randomly assigned to one of two treatment groups: probiotic Randomization. Women were randomized at the baseline visit following a 2:1 allocation ratio using the Research Electronic Data Capture (REDCapData collection. Data was collected at baseline (15–20 WG), 24–28 WG, 34–36 WG, Labor and Delivery and during monthly pill refill visits that fell outside of the main study visits. Most data collection was conducted in person with modifications made at the onset of the COVID-19 pandemic as described below. Study feasibility. The electronic health record (EHR) was screened daily to identify eligible women scheduled for a “New Obstetrics” visit. Women were tracked, and the number of women approached (by phone & in person) for enrollment and the number of women who declined was documented. Once a woman was enrolled in the study, attendance at study visits, completeness of data, and overall and treatment specific loss to follow-up/withdrawal was closely monitored and documented. To track progress of participants through the study, the Consolidated Standards of Reporting Trials (CONSORT) subject flow diagram was utilized [Health events. Health events including medication changes, iron supplementation, pregnancy-related conditions, and gastrointestinal symptoms were assessed monthly by survey and through viewing provider notes in the participant’s EHR. Study tolerability was defined Adherence monitoring. Adherence to the supplements was assessed using a Bluetooth-enabled smart pill bottle (Pillsy, Seattle, WA) and standard hand pill counts. At baseline, the smart pill bottles were paired with the participant’s smartphone by a member of the research staff, through the Pillsy phone application. Once paired, the application sent daily alerts to the subject when it was time to take the supplements (i.e., the pill bottle rang and lit up, and a secondary reminder was sent through the phone application). Data was also transmitted to a HIPAA-compliant research platform that tracked the number of times the bottle was opened with a date and timestamp. The technology allowed members of the research staff to receive daily dose-compliance information and allowed for two-way text and call communication between participants and study staff if data from the smart bottle were not transmitted. To proactively account for technological challenges related to the smart bottles, UIC IDS performed standard hand pill counts at each bottle return and recorded the number of pills taken and remaining in each bottle in a spreadsheet that was shared with research staff when the study was completed.Maternal and Cord Blood Collection and Processing. At each data collection visit, maternal venous whole blood was obtained, with a portion processed for serum, to assess maternal iron, inflammatory and hematological parameters. At delivery, a venous umbilical cord blood sample was obtained at the bedside following delivery of the placenta. All maternal and cord blood samples were processed and stored at −80 °C or sent immediately to a commercial laboratory (Quest Diagnostics, Wood Dale, IL, USA) for analysis. Complete Blood Count (CBC). CBC with differential was measured in whole maternal or cord blood by electronic cell sizing/counting/cytometry/microscopy by a local commercial lab (Quest Diagnostics, Wood Dale, IL, USA). Hb, obtained from the CBC, was used to define trimester-specific maternal IDA, with a downward correction of 0.8 g/dL for Black women [Iron Status Parameters. Serum ferritin and iron were measured from maternal and cord serum by immunoassay and spectrophotometry by a local commercial lab (Quest Diagnostics, Wood Dale, IL, USA). Normal ranges for maternal ferritin are: 2nd trimester 2–230 ng/mL and 3rd trimester 0–116 ng/mL [C-reactive Protein. High-sensitivity CRP (hs-CRP), a common clinical marker of systemic inflammation, was measured via nephelometry from maternal and cord serum by a local commercial lab (Quest Diagnostics, Wood Dale, IL, USA).Survey Interviews. Interview-administered surveys included a socio-demographics and health history questionnaire and a 24-h diet recall. The 24-h diet recall was conducted using the Nutrition Data System for Research (NDSR) software (University of Minnesota, Minneapolis, MN, USA) using a multiple-pass interview approach, to standardize collection of the data [Maternal Anthropometrics. Maternal height was measured using a fixed stadiometer (baseline only) and weight at each gestational data collection visit using a calibrated digital scale (or via EHR during the COVID-19 pandemic) and at admittance for labor and delivery (from EHR). BMI was calculated as kg/mNeonatal Characteristics. There is evidence that fetal sex, neonatal weight at delivery and gestational age at delivery can affect maternal and neonatal iron status and hematological parameters [Data Management. Research staff entered all data directly into a REDCap (Vanderbilt University, Nashville, TN, USA) data structure. Standard checks for outliers, duplicates, and other errors associated with data entry were conducted. Power and Statistical Analysis. We aimed to recruit 24 individuals, with the assumption of 80% retention, resulting in a final recruitment sample of 20. With a 2:1 allocation ratio distribution between groups, our sample size is adequate to estimate parameters (e.g., standard deviation, mean change) needed to inform the design of a future efficacy trial. Confidence intervals for feasibility proportions such as retention and percent adherence have a maximum width of 0.27 if proportions are 0.5, and 0.22 with a proportion of 0.8 [Statistical analysis was performed using Stata/BE (versions 15 and 17, College Station, TX, USA). Stata variables not normally distributed were transformed using natural log transformation, and data were presented as geometric means with confidence intervals. For data that could not be normalized with transformation, non-parametric tests were utilized. Given the feasibility nature of the study, the analysis was largely descriptive. For continuous variables, means, confidence intervals, medians, interquartile ranges, and standard deviations were reported. For categorical variables, frequencies and percentages were reported. The feasibility and supplement adherence data were reported by all randomized participants and by per protocol analysis (≥80% supplement adherence, completed the study). The maternal and cord blood hematological markers presented were from the per protocol analysis (≥80% supplement adherence, completed the study). For the maternal data, we visualized changes in the hematological and iron status parameters using “spaghetti” plots to provide a demonstration of marker changes over the gestational period. Differences between treatment groups (