Developmental Impacts of Microplastics Exposure in Early Life

The proposed pilot study will establish a new cohort of healthy, lactating women from Baltimore City and Baltimore County, Maryland. By recruiting women from these two regions, the investigators will be able to ensure that the pilot study covers a diverse geographical range with varying levels of air pollution exposure and social determinants of health (SDoH). Study participants will be 1 or 3 months postpartum (+/- 2 weeks) and report exclusive breastfeeding. The investigators will also biobank maternal stool samples for future funding applications that will additionally examine the gut microbiome and fecal metabolome.

Height, Weight, Blood Pressure, and other Anthropometric Measures: Maternal height and weight will be measured using a calibrated medical scale and a commercial stadiometer. Infant weight will be measured in duplicate on a digital scale accurate to the nearest 10 g and length will be measured in duplicate to the nearest 0.1 cm using an Infantometer and the standardized World Health Organization (WHO) approach. Infant skinfold thickness will be measured at four sites with Holtain skinfold callipers. Maternal weight will be recorded to the nearest 0.1 kg and height will be recorded to the nearest 0.1 cm. Automated blood pressure using appropriate cuffs will be used to obtain three readings of blood pressure, from which the average value will be recorded, per recommendations of the American Heart Association.

Assessment of Food Intake; Physical Activity: Dietary data will be collected using the National Cancer Institute (NCI) National Health and Nutritional Examination Survey (NHANES) Food Frequency Questionnaire. The questionnaire is designed to capture information about food intake over the prior month, and includes questions outlining consumption of fruits, vegetables, dairy products, whole grain and fiber, sugar, and red and processed meats. The questionnaire will be administered on the web with a research assistant nearby to assist with any questions. Given the investigators are interested in microplastics exposure from food, additional dietary data will be collected through the administration of Nutrition Data System for Research (NDSR) 24-hour dietary recalls which will be administered over the phone by a registered dietician or study staff under the direct supervision of a registered dietician. The investigators will ask mothers to complete 3 dietary recalls for themselves and 3 for their infant, for ideally 2 weekdays and a weekend. One dietary recall for both mother and infant will be completed during the Clinical Visit at the Clinical Research Unit (CRU). Research assistants will work together with participants to establish which days are best suited for them to complete subsequent recalls. Finally, to assess the quantity of milk consumed by infants during a single feeding, the investigators will weigh the infants at the study visit before feeding. Then mothers will be asked to breastfeed their infant. Upon completion of feeding, the investigators will re-weigh the infants to roughly capture the amount of milk consumed.

Maternal and Infant Fecal Samples: Fecal collection kits will be given to eligible participants. To avoid plastic contamination, maternal fecal collection kits will include an absorbent pad covering any plastic on the stool collection hat, gloves, a metal spoon, and glass container. For the infant stool collection, mothers will be provided with a diaper, bee's wrap; cloth, and all collection supplies that are free of plastic. The investigators will deliver these collection supplies and instructions to participants prior to their visit so that participants may collect these samples at home. The investigators will ask them to take the samples within 24 hours of the visit time to preserve the microbial composition of the stool samples. The instructions will specify that samples should be stored in the refrigerator until their visit. The investigators will provide a cooler for participants to transport the samples to the CRU. If for any reason, participants were not able to obtain these samples at home, participants will be allowed to collect them during the visit, If collected at the clinical visit, maternal and infant stool samples will be transferred into 3-4 glass vials and stored in a -4°C fridge for 24 hours prior to being stored at -80°C for MNP and gut microbiome sequencing. Fecal collection tubes utilize a fecal preservative, which prevents changes to aerobic and anaerobic bacteria that can occur during shipment. Fecal sequencing will be performed at Emory University (Dr. Doug Walker). Only de-identified samples will be sent to Dr. Walker's lab. DNA will be sequenced on the Illumina HiSeq 4000 platform. Microbial composition profiles will be predicted using MetaPhlan. Samples will be functionally mapped using HUMAnN2 to obtain abundance level of gene families from the UniProt Reference Clusters that will be further mapped to MetaCyc metabolic pathways database. Genes will be grouped in clusters of orthologous groups from EggNOG data. Gene family abundances will be grouped into broader functional categories based on annotation for the UniProt gene ontology. For the metabolomics analysis, fecal samples will be analyzed using ultra-performance liquid chromatography (UPLC) and high resolution/tandem mass spectrometry (MS/MS) by Metabolon. Compounds of exogenous, human, and microbial origin will be identified by comparison to library entries of purified standards or recurrent unknown entities. Metabolon maintains a reference library of more than 4,500 known metabolites and more than 9,000 novel metabolites. Biochemical identifications will be based on three criteria: retention index, accurate mass match to the library +/- 10 ppm, and the MS/MS forward and reverse scores between the experimental data and authentic standards.

Breast Milk Expression: Due to potential exposures from feeding breast milk from plastic bottles, all mothers will be asked to only feed their infants from the breast for 14 days prior to infant stool collection. Similar to the stool collection supplies, the investigators will provide mothers with a Haakaa Silicone Breast Pump, silicone storage container with cap, and instructions prior to the study visit. Mothers will be asked to provide a full expression of a single breast. If mothers are uncomfortable providing a full expression, study coordinators will work with mothers to determine an amount appropriate for collection. The investigators will collect a minimum of roughly 4g of breast milk. If mothers are unable to collect their breastmilk sample before the visit, mothers will be provided with a quiet, private CRU study room to pump using a Haakaa Silicone Breast Pump into a silicone storage container. Following collection, a member of our study team will aliquot measures into glass sterile tubes for storage. Following collection, all samples will be stored at -80 degrees C prior to processing and analysis.

Blood Draw: Blood draws will be collected (40mL) for microplastics analysis. Blood will be collected using a stainless steel 21G needle that is connected to a glass container under vacuum, so blood drawn directly from participant is deposited in the glass vacutainer. Glass vacutainers will have a rubber seal for storage.

Inclusion criteria

• All gender expressions
• Mothers at 1 or 3 months postpartum (± 2 weeks)
• Intent to exclusively breastfeed for ≥ 6 months
• Nulliparous and singleton births
• Receiving only breast milk from the mother, with no formula supplementation

Exclusion criteria

• Physical, mental, or cognitive disability that prevents participation; current incarceration
• Chronic conditions in mother and/or infant (e.g., heart disease, cancer, autoimmune disease, diabetes, intestinal bowel syndrome/disease), conditions diagnosed for the first time during pregnancy (e.g. gestational diabetes), neurological conditions in mother and/or infant (e.g. epilepsy, seizures), psychological conditions in mother and/or infant (e.g. bipolar disorder), or neurological/psychological conditions diagnosed in mother during or immediately after pregnancy (e.g. postpartum depression)
• Previously diagnosed with any major illness (e.g., intrauterine growth restriction, birth asphyxia, cancer) or eating disorder or previous gastric band surgery
• Maternal antibiotic use during pregnancy or the postpartum period. Infant antibiotic use at any time during life or taking medications that could affect plastic exposure or the microbiome, such as medications administered via a plastic syringe.
• Use of any breast pump except the Silicon Breast Pump for the 14 days prior to the clinical visit.
• PHQ9 score greater than or equal to 10 indicating clinically relevant symptoms of moderate or worse depression
• Infants who are intersex
• Smoking, drug use (including marijuana use), or alcohol abuse