PRenatal and Obstetric Maternal Exposures and ISlet Autoantibodies in Early Life

We will assemble a prenatal cohort where the offspring has a first degree relative (FDR) with T1D (mother, father or full sibling). In order to enroll sufficient participants, we are conducting a multi-center study at the Barbara Davis Center for Diabetes (BDC) in Aurora, CO \[coordinating center\], Mt Sinai Hospital in New York, NY, the Joslin Diabetes Center (JDC) in Boston, MA, and Ohio State University (OSU) in Columbus, OH. These sites were chosen based on a history of successful prior collaborations, access to large established populations of people with T1D (these centers are some of the largest in the United States with respect to pregnancy care for women with T1D, and also have adult and pediatric clinics), site-specific connections to obstetric practices, and a history of successful recruitment into study cohorts. In addition to the four study sites named, we have approached additional potential sites in order to ensure success with recruitment. We have identified an additional potential site at the Mayo Clinic College of Medicine under the direction of Yogish Kudva, and this site is prepared to activate should recruitment in the first year of the grant at the four sites fall short of expectations. Our goal is to assemble a pregnancy cohort of 800 during this initial funding period, and to enroll up to an additional 1,200 in phases 2 and 3 (total N=2,000).

D. Description, Risks and Justification of Procedures and Data Collection Tools:

Screening and Consent Procedures: Participants will be screened, consented (available through paper and electronic consent process) and will complete questionnaires through an online database with electronic survey capabilities (REDCap) that is HIPAA compliant and can be accessed anywhere with internet access. If needed, REDCap access can be provided in the clinic using a tablet. We have successfully used the REDCap database system for numerous studies involving survey collection and have used online screening and consenting procedures extensively in studies of adults with T1D (including pregnant women) during the Covid-19 pandemic. Study staff will be available to review the consent form with potential participants over the phone, using a HIPAA-compliant video platform such as Zoom, or in person, depending on the circumstances. Screening questions will include the history of T1D in the pregnant woman or father/sibling of the fetus, maternal age, and obstetric history for the pregnant woman, including current pregnancy dates (last menstrual period date, expected date of delivery).

Visit and Specimen Collection Schedule: Study visits and specimen collection (Table 2) will be scheduled at one of the study sites, at one time point for fathers and at four time points for mothers: first trimester (8-\<14 weeks), second trimester (14-\<28 weeks), third trimester (28+ weeks), and up to 12 weeks postpartum. Medical records will be obtained using medical record release forms for the pregnancy, delivery and post-partum visits, and for the infants' birth records. All study sites have access to electronic health records (EHR) that will allow investigators to obtain medical records for prenatal care and delivery for study participants who deliver within their hospital network, and all sites have protocols in place and experience obtaining records from outside facilities using medical record release forms. At the BDC coordinating center, we have previously collected medical records for pregnancy, delivery and infant birth records and have abstracted these data into REDCap databases. We will follow our established procedures for abstraction of obstetric and newborn records for this study. Maternal and fetal outcomes of women included in this study that are reported to BDC providers will be obtained by manual chart review of EHR charts. Maternal and fetal hospitalizations at any hospital system utilizing EPIC and for whom patients authorize sharing of records can be viewed through EPICS's interoperable sharing tool called Care Everywhere. Manual review of records will be used to obtain specific outcome measures.

All records will be collected and abstracted at the Barbara Davis Center, to maintain quality control standards and to streamline training and oversight of staff.

Pregnancy Visits: Pregnant women who are eligible for the study will be enrolled in the first trimester (before 14 weeks gestation), if at all possible, but after 8 weeks gestation to reduce the possibility of early pregnancy loss following enrollment. Fathers will be enrolled at the same time and will be asked to complete their study visit (including a blood sample of up to 33 ml or 2 1/4 tablespoons of blood) at the same time as the woman's first pregnancy visit, if at all possible. Women who are already in the second trimester (up to 24 weeks) when approached about the study will be enrolled and will complete second and third trimester visits and the post-partum visit. Routine pregnancy visit data, including weight and blood pressure will be collected in the clinic or from the medical record. Routine pregnancy visit data, including weight and blood pressure, routine laboratory tests (including HbA1c for women with T1D, lipids, etc) will be collected from the clinic or from the medical record. Study visit timing and procedures are shown in Table 3 below.

Specimen Collection: Samples will be collected at each study visit at the participating clinic location. External centers will ship all samples to the BDC for storage. Sample collection will be standardized in our Manual of Operations to ensure consistent collection at all sites. Further, sample collection, timing and types have been carefully designed to be similar to the collection completed in existing cohorts, in particular the ENDIA study, to allow for comparison of results across studies. However, timing of trimester-based and post-partum visits are determined by standard of care in our clinics and may not align exactly with the ENDIA study. For example, women typically complete a post-partum visit at 4-6 weeks in our clinical centers, and so we will schedule our post-partum data and sample collection at the same time, to minimize participant burden. In the ENDIA study, mothers underwent a post-partum visit at 3 months.

A sample of up to 33 ml or 2 ¼ tablespoons of blood will be obtained by phlebotomy. Plasma will be collected in EDTA tubes, kept on ice and separated by centrifugation. Serum will be collected in serum separator tubes with gel and separated by centrifugation after being allowed to clot. Serum and plasma will be aliquotted into 2ml cryovials and stored at - 80° C. Buffy coats will be stored at each visit for future DNA and methylation studies. Blood RNA samples will be collected using Tempus™ tubes, which allow for stabilization and isolation of total RNA from whole blood for gene expression analysis. These samples can be stored for up to five days at room temperature, and then frozen at 4°C. Optional vaginal swabs will be self-collected by participants using a CultureSwab transport system. Optional stool samples will be self-collected by participants using a commercially available stool collection kit such as the OMNIgene\_GUT.

Questionnaire Collection: Participants will be asked to complete questionnaires online using the REDCap database program for each visit, to reduce the time needed to complete the in person appointments for study participants completing visits at the clinics, and to streamline data collection/entry procedures and reduce data entry errors. Questionnaires will include dietary intake using a validated food frequency questionnaire43, tobacco and alcohol using validated questionnaires44,45, and we will collect screening questionnaires addressing general \[Patient Health Questionnaire-8\]46 and perinatal \[Edinburgh Postnatal Depression Scale\]47 depression. Women who screen positive (a PHQ8 score of 14 or higher, or an Edinburgh Postnatal Depression score of 10 or higher) on either of these questionnaires will be referred to their regular provider for further evaluation.

Among mothers and fathers with T1D, assessments of the T1D distress scale (T1-DDS), diabetes self-management and hypoglycemia fear will be assessed using validated instruments. Fathers with T1D will complete the questionnaires at a single visit, whereas mothers with T1D will be asked to complete these questionnaires at each study visit (once per trimester, and post-partum). Diabetes care records, including HbA1cs and other laboratory tests (lipids, urinary albumin to creatinine ratio, serum creatinine), retinopathy exams and examination records will be requested for the year prior to enrollment for determination of complications and glycemic control.

Pregnancy history and outcomes: We will collect data on maternal and fetal outcomes of the pregnancy with medical records; we will obtain gestational age at delivery, infant birth weight, delivery complications, and maternal and fetal outcomes by obtaining records for each hospital admission for labor and delivery. As a backup for these hospital records and in case we are unable to access medical records (such as if a woman moves outside of our electronic health record area and we can't obtain records using a medical record release), we will collect the same data using a survey we developed for one of our previous pregnancy studies. It inquires about delivery date, gestational age, infant birth weight and length, living status of the infant, infant sex, preterm labor, type of delivery, 16 specific pregnancy complications, 15 specific labor complications, 5 specific neonatal complications, and admission to the neonatal intensive care unit (NICU). Breastfeeding practices will be collected at the post-partum visit. We will also collect data through a medical chart review for health information prior to pregnancy for women with T1D (blood pressure, kidney function, retinopathy, height and weight). We will verify gestational dating by obtaining records of ultrasounds performed for dating.

Glycemic factors among women with T1D: HbA1c and glucose values (CGM and glucose meter downloads), current insulin dosing and insulin delivery method are collected during routine pregnancy visits and will be collected from the medical record. We will also access data downloaded from CGM devices (for those women who use a CGM regularly) during routine pregnancy visits to evaluate indices of glycemic variability such as time spent in pertinent glucose ranges (time in range: 63-140 mg/dL \[pregnancy\] and 70-180 mg/dL \[post-partum\], hypoglycemia: \<63 mg/dL \[pregnancy\] and \<70 mg/dL \[post-partum\], and hyperglycemia: \>140 mg/dL \[pregnancy\] and \>180 mg/dL \[post-partum\]50, coefficient of variation, and SD).

Cord blood and stool collection: Collection of cord blood up to 20 ml will be prioritized, since it is an ideal sample for examining the DNA methylation in the infant at birth and before exposure to the outside environment, including breastfeeding or bottle feeding, exposure to microbes and other environmental factors, as a contrast to later samples obtained during early life. Collecting cord blood has been accomplished in our prior studies through agreements with screening hospitals, and through the use of cord blood collection kits sent to the mother for obtaining and mailing back the samples. Given that women will be giving birth at any number of hospitals nationally in this cohort, we will plan to send/give cord blood collection kits to the pregnant mothers with detailed instructions for collection and return of the samples, using our established protocol. In the case that cord blood collection isn't possible, we will include a kit for collecting a heel stick sample of up to 1ml of blood as soon as possible after birth (preferably within a week of delivery). We also plan to give women stool kits for collection of a stool/meconium sample soon after delivery.

Screening