Study population
The study subjects in the current analysis were from the Shanghai Birth Cohort, a large prospective study in Shanghai, China, from 2013 to 2016 [23]. The inclusion criteria were couples aged 20 years or older, married, seeking preconception care or in early pregnancy, with spontaneous conception, at least one of them being registered residents in Shanghai with no plan to move out of Shanghai for two years after enrollment. The participants provided an informed consent before enrollment, and were interviewed by trained staffs to complete structured questionnaires on demographic characteristics, previous reproductive and medical history and lifestyle factors. They also provided blood samples voluntarily at the recruitment visit. Clinically relevant characteristics of the current pregnancy such as pregnancy complications, chronic diseases during current pregnancy, number of fetus, infant sex and gestational age at birth were extracted from medical records. An ethical approval was obtained from the Ethical Committee of the Xinhua Hospital affiliated to the Shanghai Jiao Tong University School of Medicine.
For the current analysis, a total of 3242 women who provided blood samples in early pregnancy (gestational age, median (interquartile range; IQR): 15 (13 - 16) weeks) and delivered a singleton live birth were eligible. Births with missing information on gestational age at birth, gestational age < 20 weeks or > 42 weeks were excluded (n = 289). Mothers or infants with missing information on maternal age at enrollment, pre-pregnancy body mass index (BMI), parental educational level, parity, chronic diseases during pregnancy or infant sex were excluded (n = 104). Finally, 2849 mother-infant pairs were included for analysis.
PFAS measurement
Blood samples were taken during early pregnancy and immediately centrifuged and frozen at −80 °C. A detailed analytical method has been described elsewhere [24]. In brief, PFAS concentrations were measured in 100 μl plasma using high-performance liquid chromate graphy/tandem mass spectrometry (HPLC/MS-MS; Agilent1290–6490, Agilent Technologies Inc., USA). The intra- and inter-assay coefficients of variation were both below 10%. The limit of detection (LOD) was 0.09 ng/mL for PFOS and PFOA, 0.05 ng/mL for perfluorododecanoic acid (PFDoA), 0.03 ng/mL for perfluoroheptanoic acid (PFHpA), 0.02 ng/mL for PFDA, PFNA, perfluoroundecanoic acid (PFUA) and perfluorohexanesulfonate (PFHxS), 0.12 ng/mL for perfluorooctane sulfonamide (PFOSA), and 0.009 ng/mL for PFBS.
Outcome
The main outcome of this study was PTB, which was defined as birth between 21 and 36 completed weeks’ gestation. Spontaneous PTB referred to spontaneous onset labor and preterm premature rupture of the membranes (PPROM) irrespective of mode of delivery (vaginal or cesarean section), while clinically indicated PTB was defined as PTB for preeclampsia, fetal stress, placenta previa and other maternal, fetal or placenta indications[3].
Covariates
Demographic characteristics included maternal age (< 30, 30 - 34, ≥ 35 years old), parental education levels (≤ 12, > 12 years), smoking during pregnancy (no, yes, unknown), gestational age at blood collection. Obstetric and medical covariates included pre-pregnancy BMI (< 18.5, 18.5 - 24.9, ≥ 25 kg/m2), parity (0, ≥ 1), pregnancy complicated by chronic diseases (no, yes), and infant sex (female, male). Chronic diseases included heart, kidney or liver disease, diabetes mellitus, chronic hypertension disorders, epilepsy, malnutrition and anemia.
Analysis
Distributions of demographic and pregnancy related characteristics were showed as numbers and percentages. PFAS with concentrations below the LOD were assigned LOD/ . The distribution of PFAS in early pregnancy was presented as medians and interquartile ranges. Multivariable restricted cubic spline model was used to examine potential non-linear relationships between PFAS and gestational age (in weeks) at birth, according to the ln-transformed PFAS at three knots of 10th, 50th, 90th percentile. Univariable and multivariable linear regression analyses were used to evaluate the associations of ln-transformed PFAS with gestational age (in weeks) at birth. Simple and multiple logistic regression analyses were applied to estimate odds ratios (ORs) and 95% confidence intervals (CIs) of the overall PTB, spontaneous PTB and clinically indicated PTB. In the logistic regression models, PFAS were entered as continuous variables (ln-transformed) and in tertile (untransformed) with the lowest tertile served as the reference group. Based on the directed acyclic graphs and existing literature, covariates were included in all adjusted models, including maternal age (years), pre-pregnancy BMI (kg/m2), parental education levels (≤ 12, > 12 years), parity (0, ≥ 1), chronic diseases (no, yes), infant sex (female, male) and gestational week at blood collection. Because only few mothers (n = 9, 0.3%) smoked during early pregnancy, maternal smoking status was not included in the adjusted models.
To investigate the potential modification of infant sex, we performed a stratified analysis. To examine whether the associations differed among women who were multiparous, had preterm labor history, or complicated by chronic diseases, we carried out several subgroup analyses, restricting to women without these conditions, respectively.
Spearman’s rank correlation coefficient was used to estimate the correlations between each pair of PFAS. As multicollinearity between PFAS may bias the estimated effects, we conducted linear regression or logistic regression models with mutual adjustment of the correlated PFAS (ρ > 0.4).
All analyses were carried out using SAS version 9.4 (IBM SAS Institute Inc, Cary, NC) and R software (R version 3.6.1). Results at p < 0.05 were thought to be statistically significant.