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.