Oxygen tension regulates the miRNA profile and bioactivity of exosomes released from extravillous trophoblast cells – Liquid biopsies for monitoring complications of pregnancy.

Cell culture

All experimental procedures were conducted within an ISO17025 accredited (National Association of Testing Authorities, Australia) research facility. All data was recorded within a 21 Code of Federal Regulation (CFR) part 11 compliant electronic laboratory notebook (Lab Archives, Carlsbad, CA 92008, USA). The HTR-8/SVneo cell line was kindly donated by Dr. Charles H. Graham (Queen's University, Ontario, Canada). HTR-8/SVneo was established by the transfection of trophoblast cells isolated from first trimester villous explants and containing a gene encoding the Simian virus 40 large T antigen to immortalize them [ 13 ]. HTR-8/SVneo is commonly used as an EVT model and HTR-8/SVneo authentication of human trophoblast cell line was performed with a STR DNA Profiling Analysis ( S1 Fig ). HTR-8/SVneo cells were maintained in phenol red-free RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% non-essential amino acids, 1 mM sodium pyruvate, 100 U/mL penicillin, and 100 mg/mL streptomycin. Cultures were maintained at 37°C and humidified under an atmosphere of 5% CO 2 -balanced N 2 and either 8% or 1% O 2 in an automated PROOX 110-scaled hypoxia chamber (BioSpherics ™ , Lacona, NY, USA). Cells were cultured in RPMI 1640 medium supplemented with 10% FBS-exosomes depleted for 48h before exosome isolation. Cells were sub-cultured with dissociation media, TrypLETM Express (Life technologies, USA). Cellular viability was determined by Trypan Blue exclusion solution and Countess ® Automated cell counter (Life Technologies, USA).

Patients

A case control study design was used to establish pregnancy-associated changes in exosomal miRNA present in maternal plasma obtained from normal, preeclampsia and preterm birth pregnancies. Human plasma samples were obtained in accordance with the Declaration of Helsinki. This study was approved by the ethics committee of The University of Queensland (HREC/09/QRBW/14) and Ochsner Medical Foundation (USA). Women were recruited with informed written consent at the Ochsner Baptist Medical Center (New Orleans, USA). Samples were obtained before 20 weeks of gestation and classified according to the pregnancy outcomes in normal, PE or SPTB. PE was defined as new-onset hypertension [(BP) blood pressure ≥140/90mmHg on two separate occasions at least 6h apart or BP ≥160/110mmHg)] and proteinuria (>300mg/24h) after 20 weeks of gestation in previously normotensive women, according to the International Society for the Study of Hypertension in Pregnancy [ 14 ]. SPTB was defined as a birth before gestational week 37 + 0. The clinical characteristics of the patients included in this study are presented in Table 1 . 10.1371/journal.pone.0174514.t001 Table 1 Clinical characteristics of patients. Variables Normal (n = 6) PE (n = 6) SPTB (n = 6) Maternal age (years) 31 ± 2.9 (29–35) 32 ± 4.3 (28–33) 30 ± 5.4 (26–33) Height (cm) 165 ± 0.6 (160–170) 159 ± 0.7 (155–170) 160 ± 0.7 (150–165) Weight (Kg) 61 ± 5.3 (55–68) 63 ± 5.3 (55–70) 61 ± 4.6 (55–69) BMI (Kg/m 2 ) 23 ± 1.5 (20–26) 25 ± 1.3 (20–29) 24 ± 1.8 (20–27) Systolic/diastolic blood pressure (mm Hg) 109/65 ± 6.22/9.12 (90-120/50-81) 155/69 ± 10/8 * (>140/90) 111/67 ± 10/7 (90-120/50-80) Gestational age at sample collection (weeks) 17 ± 3.5 (15–18) 16 ± 2.6 (15–18) 16 ± 2.8 (15–18) Gestational age at delivery (weeks) 40 ± 1.2 (37–42) 38 ± 0.9 (37–39) 35 ± 1.1 (34–36) Data are presented as mean ± SD (range). PE = preeclampsia; SPTB = spontaneous preterm birth. *p