Elevated third trimester corticosteroid levels are associated with fewer offspring infections – Scientific Reports

Cohort descriptions

VDAART was a randomized, double-blind, parallel-design study conducted at three study sites across the United States that preferentially recruited asthmatic mothers (ClinicalTrials.gov identifier: NCT00920621)¹⁷. VDAART recruited pregnant women at 10–18 GW and randomized them to Vitamin D supplementation at 4000 IU/day or placebo; all women received 400 IU/day vitamin D supplementation as part of usual pregnancy care. Blood samples were collected in EDTA tubes during enrollment between 10 and 18 GW and again between 32 and 38 GW; plasma was separated through centrifugation at 2000 RPM at 4 °C, and samples were stored at −80 °C until metabolomic profiling. For simplicity, throughout the manuscript, the earlier VDAART pregnancy time point (10–18 GW) will be referred to as “T1”, and the later VDAART pregnancy time point (32–38 GW) will be referred to as “T3”.

COPSAC was a single-center, pre-birth cohort study, as described previously¹⁸. As part of the COPSAC study, mothers were randomized to vitamin D₃ supplementation of 2400 IU/day vs. placebo; all women received 400 IU/day of vitamin D₃ as part of usual pregnancy care. Blood samples were collected in EDTA tubes from mothers during the 22–26 GW enrollment visit; plasma was separated and stored at -80 °C until processing¹⁹. Since this pregnancy time point occurs between the two VDAART time points, the COPSAC pregnancy period of 22–26 GW will be referred to as “T2” for simplicity.

Infection proneness, asthma, and lung function outcomes in offspring

Respiratory infections in VDAART offspring were recorded between the birth of the child and age 6 years using standardized quarterly questionnaires completed by caregivers. In order to match the COPSAC reporting period, infections occurring only between birth and age 3 years were evaluated in this study. Respiratory infections in COPSAC offspring were recorded during scheduled clinical visits every 6 months to 1 year through parent report, beginning at birth until age 3 years. For both cohorts, the infection proneness outcome is a continuous variable representing the count of total reported respiratory infections reported, including upper respiratory infections, lower respiratory infections, colds, and ear infections. Asthma status in VDAART and COPSAC was recorded as positive if the child was diagnosed by a physician with asthma and/or recurrent wheeze any time between birth and age 6 years. Lung function spirometry measurements were collected in both cohorts at age 6 years, including forced expiratory volume in the first second (FEV₁), forced vital capacity (FVC) and the ratio of FEV₁/FVC; further details of spirometry measurement are available in Supplemental Methods S1.

Steroid metabolite measurement by UPLC-MS/MS

Metabolomic profiles for VDAART and COPSAC plasma samples were generated by Metabolon, Inc. (NC, USA) using ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry (UPLC–MS/MS). A total of 16 steroid metabolites were included in analysis, including 14 androgenic and 2 corticosteroid metabolites. Of these, 15 were Tier 1 assignments by Metabolon, with full structure identification matched to a standard. One androgenic steroid metabolite met putative Tier 2 identification by matching to a literature database, and is denoted with a superscript hash sign in Tables. Details of UPLC-MS/MS methods are available in Supplemental Methods S1.

Metabolite associations with childhood outcomes

Regression analyses estimated associations between steroid metabolites and childhood outcomes at each of the three maternal time points using Poisson (infection proneness), logistic (asthma), or linear (lung function) regression models. Potential confounding factors were selected based on previous research (Fig. 1)⁶. VDAART models were adjusted for maternal age, race, ethnicity, pre-pregnancy BMI, vitamin D level (nanograms of 25 hydroxyvitamin D [25(OH)D] per milliliter of blood), maternal education status, birth order of child, and smoking during pregnancy; an additional variable for gestational days at collection was included for T1 only, as this time point spanned a longer eight-week period. COPSAC was homogenous with respect to race and ethnicity, so models used the same covariates as VDAART models except race and ethnicity. A restricted analysis of non-asthmatics mothers was also conducted for the infection proneness outcome to control for potential confounding by asthma status or asthma medication use (details in Supplemental Methods S1). Analyses were conducted using the stats package in R version 4.0.3²⁰, and incidence rate ratios (IRRs), odds ratios (ORs), linear regression coefficients, and P-values are reported, as appropriate. Correction for false discovery rate (FDR) was performed using the Benjamini–Hochberg Procedure.

Mediation of the relationships between maternal steroids and infection proneness was assessed for the following potential mediators: gestational age at delivery, pregnancy-induced hypertension, gestational diabetes, pre-term labor, delivery mode, maternal asthma, and child daycare attendance during the first 3 years of life. Mediation analyses were performed using the stats²⁰ and mediation²¹ packages in R 4.0.3.

Ethics approval and consent to participate

All portions of this study were conducted in accordance with the Declaration of Helsinki. Local institutional review boards approved the protocol, and written informed consent was obtained from all individuals. The institutional review boards at each participating VDAART Clinical Center and the Data Coordinating Center at Brigham and Women’s Hospital approved protocols of the trial. This project was additionally approved by Brigham and Women’s Hospital IRB protocol 2018P000478. The COPSAC study was conducted in accordance with the Declaration of Helsinki and was approved by the Copenhagen Ethics Committee (KF 01-289/96) and the Danish Data Protection Agency (2008-41-1754).