Collection of blood samples will be according to the recommended order of draw (Multimedia Appendix 1): the Vacuette serum tube (silica-coated clot activator and polymer gel; Greiner Bio-One) for use in routine clinical chemistry and hormone tests, serology, and immunohematology; the Vacuette heparin plasma tube (lithium heparin coated) used for clinical biochemistry tests; Vacuette ethylenediaminetetraacetic acid (EDTA) tubes (containing K2 EDTA anticoagulant) used for hematological procedures, such as complete blood counts and blood typing and plasma isolation, to allow for multiomics analyses; and the PAXgene blood RNA tube (containing 6.9 mL of RNA stabilization additive; BD Biosciences) used to stabilize intracellular RNA to yield accurate and reproducible gene expression data [23]. Maternal blood samples will be collected at 14-, 20-, 28-, 36-, and 40-week appointments, as well as 6 weeks postpartum. Partner blood samples will be collected at the 20-week appointment, and cord blood will be collected at birth. Adult blood samples to be collected include 8 mL in one 8 mL Vacuette serum tube; 4 mL in one 4 mL Vacuette heparin plasma tube; 18 mL in two 9 mL Vacuette K2 EDTA tubes; and 4 mL of cord blood in one 4 mL Vacuette EDTA tube. The buffy coat, containing white blood cells and platelets, an important source of genomic DNA that can withstand long-term storage [24], will be extracted from centrifuged Vacuette EDTA tubes. In addition to these tubes, 2.5 mL (draw volume) in 1 PAXgene blood RNA tube will be collected for maternal blood samples at 14-, 20-, and 28-weeks’ gestation. Thus, each family will have the potential to donate blood that totals up to 68 aliquots within their first year of the study that can be used for traditional and nontraditional -omics techniques.

Using a tourniquet on the arm above the cubital fossa, a research nurse will locate a suitable vein and clean the site with an alcohol swab. Samples will be collected via a 21G sterile luer and holder with a retractable needle, commonly used during venipuncture as the smaller gauge reduces discomfort and prevents hemolysis. Once all tubes are filled and the needle removed and retracted for safe disposable, a sterile injection pressure pad will be applied to the collection site.

The placenta will be placed in a sterile specimen dish postbirth. If umbilical cord blood gas analysis is required as part of clinical care, it will be obtained prior to research samples. A large cord vein will be located and cleaned using an alcohol swab to remove any maternal blood. An 18G needle will then be inserted into the vein, and a 10 mL syringe will be used to draw a blood sample, which will be put into a 4 mL EDTA tube.

The subsequent sections will discuss the methodology of processing, separation, and storage of plasma, serum, and the buffy coat from the EDTA, serum, lithium heparin, and PAXgene blood tubes (Figure 2).

Samples will be inverted 5-10 times to ensure adequate mixing of blood with the EDTA anticoagulant [25] and centrifuged under the following conditions: 10 minutes at 500 ×g at 4 °C, with an acceleration of 9 and a deceleration of 3. All but approximately 0.2 mL of plasma (reserved for the buffy coat; Figure 3) will be aliquoted into one 15 mL Falcon tube and subjected to a second centrifugation step for 10 minutes at 2000 ×g at 4 °C, with an acceleration of 9 and a deceleration of 9. Plasma will be aliquoted into a new sterile 15 mL Falcon tube (Corning) and centrifuged again according to the same conditions as the previous spin and the pellet discarded. The remaining plasma will then be aliquoted: 1.5 mL plasma into one 1.5 mL low-binding Eppendorf tube, with the remaining plasma evenly distributed among four 2 mL cryovial tubes. For 36-week appointments, 0.2 mL plasma will be transferred into two 2 mL cryovial tubes containing 0.6 mL of DNA/RNA Shield solution prior to the plasma being distributed into the four 2 mL cryovial tubes. Cord blood samples will be subjected to the same centrifugation conditions; however, a smaller quantity of plasma will be obtained. Approximately 0.5 mL of plasma will be placed into one 1.5 mL low-binding Eppendorf tube and two 2 mL cryovial tubes. When extracting plasma, approximately 0.2 mL will be reserved (in each tube) for the buffy coat. Aliquots will be stored at –80 °C until required for analysis, and the OpenSpecimen database will be updated to include these aliquots.

A sterile 3 mL Pasteur pipette will be used to aliquot the buffy coat from both 9 mL Vacuette EDTA tubes (one 4 mL Vacuette EDTA tube for cord blood), along with the remaining 0.2 mL of plasma and a small portion of red blood cells into one sterile 2 mL cryovial tube. The tubes will be stored frozen at –80 °C until required for analysis, and OpenSpecimen will be updated to include these aliquots.

Samples will be inverted 5-10 times to activate the silica clotting agent [25] and then kept upright at room temperature for 20 minutes to allow clot formation. Afterward, the samples will be centrifuged under the following conditions: 10 minutes at 2000 ×g at 4 °C, with an acceleration of 9 and a deceleration of 9. The serum will be subsequently aliquoted: 1 mL into four 2 mL cryovial tubes and, for 14 weeks’ gestation only, approximately 0.1 mL into one 1.5 mL low-binding Eppendorf tube. Aliquots will be stored frozen at –80 °C until required for analysis, and OpenSpecimen will be updated to include these aliquots.

Samples will be inverted 5-10 times to ensure adequate mixing of blood with the heparin anticoagulant [25] and centrifuged under the following conditions: 10 minutes at 2000 ×g at 4 °C, with an acceleration of 9 and a deceleration of 9. All the plasma will be aliquoted into one 15 mL Falcon tube and subjected to a second centrifugation spin under the same conditions as the previous spin. All the plasma will then be aliquoted into one 2 mL cryovial tube or divided evenly between two 2 mL cryovial tubes if there is >2 mL of plasma. Aliquots will be stored at –80 °C until required for analysis, and OpenSpecimen will be updated to include these aliquots.

Samples will be kept upright at room temperature for a minimum of 2 hours before being stored at –30 °C overnight, and OpenSpecimen will be updated to include the samples. The following morning, the samples will be transferred from –30 °C to permanent storage at –80 °C until required for analysis.

Maternal saliva samples will be self-collected at home on the morning of the 20- and 36-week appointments. Partners will provide a saliva sample at 28 weeks. Participants will be instructed to collect samples within 20 minutes of waking up, before food, drink, or other substances are consumed and before teeth are brushed, to enable assessment of baseline levels of biomarkers and stress hormones, such as cortisol. To maximize the collection amount, participants will be instructed to let saliva accumulate in the mouth without swallowing for 1 minute and then to passively drool the accumulated saliva into a sterile 5 mL screw-cap collection tube (Sarstedt Australia). Samples will be processed by centrifugation in 5 mL collection tubes for 10 minutes at 2000 ×g at 4 °C prior to being placed into two 2 mL cryotubes using a sterile 3 mL Pasteur pipette. Duplicate aliquots will be implemented to minimize the need for freeze/thaw and provide an alternative or complimentary examination to blood samples. Samples will be routinely kept at –80 °C for long-term storage [15].

Maternal buccal swabs will be collected at 20 and 36 weeks’ gestation and 6 weeks postpartum; paternal samples at 20 weeks; and infant samples at birth, 6 weeks postpartum, and 6 months postpartum. Prior to collection, adult participants will be asked to refrain from eating, drinking, chewing gum, brushing teeth, or smoking for at least 30 minutes. Participants will be instructed to remove a sterile swab from the tube, ensuring the tip does not touch any surfaces, and rub the swab on both sides of their inner cheek for 60 seconds, rotating the end several times to collect skin cells. Once the collection is completed, the participants will be instructed to remove the swab, being careful the swab tip does not touch the teeth, lips, or any other surface, then place the swab directly into the collection tube, and close the lid firmly. Collection of infant buccal swabs will be completed by a research midwife or nurse.

Analysis of nail samples has proven effective for a range of environmental contaminants, including heavy metals, such as mercury and arsenic; endocrine disrupting chemicals, such as bisphenol A; and other industrial chemicals, such as chlorinated paraffins and per- and polyfluoroalkyl substances (PFASs) [67-71]. Thus far, studies that have used toenail sample analysis in infants have primarily focused on heavy metal exposure, correlating detected levels with maternal dietary exposure from 8 weeks postdelivery through 36 weeks from maternal and infant toenails at prenatal and postpartum time points. Collecting toenail samples to analyze environmental contaminant exposure is a promising yet underused tool, with the detection of an expansive range of harmful contaminants yet to be addressed in the context of fetal exposure and gestational cohorts.

Participants will be directed to self-collect toenail clippings after a bath or shower, with cleaned clippers or scissors (maternal and partner samples at 20 weeks’ gestation and maternal and infant samples 6 weeks postpartum). Participants will be instructed to cut toenails as close to the nail bed as comfortable over a clean piece of paper or tissue, with at least 2-3 mm of clippings from each toenail being obtained. The tissue or paper will be folded at the edge of each side to enable ease of transfer into a provided self-sealing envelope to be stored in a dry place at room temperature.

Similar to toenails, hair samples can provide information about toxicology, exposure to heavy metals, harmful chemicals in the environment, and alcohol and illicit substance use. Hair may also be used as a biomarker in chronic stress and certain pathological conditions [72]. As evidenced by the assessment of cortisol concentrations, 3 cm of hair growth from the scalp represents retrospective activity of the hypothalamic-pituitary-adrenal (HPA) axis in the preceding 3 months [73]. A small amount of hair cut close to the scalp is an easily accessible, noninvasive sample that can be obtained with high compliance from many populations [74,75].

Maternal and partner hair samples will be requested at 20 weeks’ gestation and maternal and infant samples 6 weeks postpartum. Hair will be parted with a comb horizontally on the crown of the head, and a 2-3 mm section of hair below will be sectioned off and, aiming for at least 3 cm of length, cut with sharp hair-cutting shears as close to the scalp as possible. For hair less than 3 cm in length, a 4-5 mm section of hair will be collected. The cut end of the hair will be fastened with medical tape. Samples will be stored, flat, in foil within an envelope and stored in a dry, dark place at room temperature. Hair samples can be stored in this way for several years, with little to no degradation [73-75].

Placental weight and processing time will be recorded. As the membrane must be repositioned or removed prior to placental villous core sampling, the fetal membrane will be collected first. The site of rupture will be ascertained and a cut made posteriorly to collect a 2 × 3 cm² section, which will immediately be washed in ice-cold phosphate-buffered saline (PBS) tablets (Gibco) at pH 7.45) and then dabbed on Kimwipes (Kimtech) to remove excess PBS prior to being snap-frozen in 2 mL cryotubes. For subsequent storage, samples will be frozen at –80 °C until required for analysis. In addition to frozen samples, a section of rolled membrane approximately 2 × 5 cm² will be obtained from the rupture site. This membrane strip will be washed in ice-cold PBS prior to placing the amnion face down on a Kimwipe, and forceps will be used to gently roll the sample. Each edge of the membrane roll will then be subjected to trimming with scissors and placed into an immunohistology embedding cassette (TechnoPlas). The cassette containing the fetal membrane roll will then be placed into a 70 mL specimen pot and subsequently washed 3 times in PBS and placed on a rocker in formalin at 4 °C overnight. Following fixation, formalin will be removed and replaced with a PBS/sodium azide solution (0.1M PBS with 0.05% sodium azide) to prevent bacterial growth for long-term storage. Fixed samples will be stored for the long term at 4 °C.

The number of samples obtained from the placenta will be determined by the weight of the placenta and whether clinical histopathology examination has been requested. Placentas<500 g will have a maximum of 6 sample cores, while in placentas>500 g, up to 8 cores may be collected. This is to ensure that all placental integrity can be maintained upon returning the placenta to histopathology for subsequent clinical macro- and microscopic evaluation. Core samples will be obtained numerically from each of the 4 quadrants (north, south, east, and west) of the placenta (Figure 4) in a process adapted from Burton et al [76]. Cores will be gathered by an 8 mm biopsy punch (Kai) and immediately placed in a petri dish filled with ice-cold PBS, ensuring that the samples are washed gently and thoroughly to minimize maternal blood carryover. Samples will be transferred to a new petri dish containing ice-cold PBS, where scissors will be used to exclude the fetal and maternal membranes. Following this, 4-6 samples (depending on placental weight) will be placed into 1 screw-cap 2 mL cryotube and snap-frozen in liquid nitrogen. Due to limited samples, tissue will not be homogenized immediately prior to being frozen, although we will form a representative cross section once the samples are frozen within the tubes. Tubes will be stored frozen at –80 °C until required for analysis. The remaining core samples will be placed in immunohistology embedding cassettes and fixed and stored, as described before. In addition to whole placental samples, individual biopsies will be obtained from the initial core sites via Klini Tischler (3 × 7 mm² jaw) biopsy tool. Samples will be rinsed in PBS, dabbed dry, and placed in uniquely labeled and barcoded individual Eppendorf tubes (1.5 mL) to represent their respective quarter.