Ethics approval and consent to participate
This is a prospective cohort study conducted on pregnant women. The study protocol was approved by the research institute’s committee of human research in the Second Affiliated Hospital of Shantou University Medical College (NO.2016027), and abided by the standards of the Declaration of Helsinki. All methods were carried out in accordance with relevant guidelines and regulations. Informed consent was obtained from pregnancy women.
Participants
We collected data of pregnant women in the obstetrics clinic from January 2018 to January 2020 for prospective analysis. The inclusion criteria of pregnant women were as follows: (1) singleton pregnancy, (2) intended to have regular antenatal care and give birth in our hospital, and (3) be able to understand the relevant scale options in this subject. The pregnant women were excluded from this study if they and the baby’s father met the following criteria: (1) had thyroid, liver, kidney, lung or heart disease before and after pregnancy, (2) had depression, anxiety disorder, somnipathy, schizophrenia, mania, dissociative personality disorder, and other mental disorders prior to pregnancy, and (3) pregnancy was by assisted reproduction.
Questionnaire survey
Participants were required to complete a questionnaire, at the first visit for antenatal care, which was used to collect variables including the age of the pregnant women, pre-pregnancy body mass index (BMI), education level, economic level, history of adverse pregnancy, pregnancy complications, and life style (smoking, drink, diet, sleep).
Data collection
Data collection was performed after delivery, including age of mother, BMI, exposure to passive smoking, adverse pregnancy history, education level, economic level, vegetarianism, pregnancy anemia, hypertensive disorder complicating pregnancy (HDCP), pregnancy diabetes, pregnancy hypothyroidism, placenta previa, uterine infection, method of delivery, preterm birth, gestational age, birth weight, and gender. Adverse pregnancy history included spontaneous abortion, induced abortion, stillbirth, and preterm birth (12). Education level was divided into with and without a college education. Economic level was divided into two levels according to the minimum taxable income, which is 5000 yuan in China. Diagnosis of pregnancy anemia (13), hypertensive disorder complicating pregnancy (HDCP) (14), pregnancy diabetes (15), uterine infection (16), and placenta previa (17) were according to the diagnosis guidance.
Assessment of maternal depression in the second trimester
Pregnancy women were asked to fill out a self-rating depression scale (SDS) during the second trimester and third trimester, respectively. The SDS consisted of 20 items, including two items of psycho-emotional symptoms, eight items of physical disorders, two items of psychomotor disorders, and eight items of depressive psychological disorders. The score for each item ranged from 1 to 4, and the total score was multiplied by 1.25 to obtain a standard SDS scale. A standard score ≥ 50 indicates depression (18). The total reliability coefficient of the SDS was 0.784 (Cronbach’s alpha) in Chinese women of rural areas, and it has been proven to be a valid and efficient tool for screening depression in the Chinese population (18).
Diagnosis of preterm birth and SGA birth
Preterm birth is a delivery before 37 weeks gestational age. The diagnosis of SGA is a birth weight of infants less than the 10th percentile for gestational age, using the Chinese neonatal birth weight curve (19).
Placenta sample collection
Approximately 1 g tissue from the maternal side of the placenta in each participant was obtained immediately after delivery. Samples of placental parenchyma were carefully dissected by trained research assistants to assure the maternal decidua was separated from the sample, from which the DNA extracted was of fetal origin. The samples were snap-frozen in liquid nitrogen, and after at least 1 hour later, stored in sample tubes at 80℃ for further examination.
Selection of placental samples for methylation detection
Methylation levels of the promoter region of the CRH and DIO3 genes were assessed by next generation sequencing-based bisulfite sequencing PCR (NGS-BSP). Because NGS-BSP is expensive, we used a nested case-control study to select specimens for examination. There were 97 pregnant women with depression in the second trimester in this study. We selected placental samples from these pregnant women and were matched 1:1 with samples from pregnant women without depression but the same maternal age, BMI, educational level, economic level, adverse pregnancy history, HDCP, pregnancy diabetes, uterine infection, and method of delivery.
Assessment of methylation levels of the promoter regions of placental CRH and DIO3 genes by NGS-BSP
Methylation was assessed by NGS-BSP. BSP primers were designed using the online MethPrimer website. DNA samples were extracted using the QI Amp DNA Mini Kit (Qiagen, Inc.). Purified DNA was quantified using an ND-1000 spectrophotometer (Nanodrop), and DNA samples (1µg) were bisulfite-modified using an EZ DNA Methylation Kit (Zymo Research). For each sample, BSP products of target genes were generated, pooled equally, and subjected to adaptor ligation. Barcoded libraries from all samples were sequenced on the Illumina HiSeq platform using a paired-end 150 bp strategy.
Statistical analysis
For continuous variables, the Shapiro-Wilk test was used to determine the normal distribution of the continuous variables, and the Wilcoxon-Mann-Whitney U-test was conducted for skewed distributions (presented as the median and the interquartile range). Descriptive statistics for categorical variables were reported as frequency (percentage) and compared using the Pearson chi-square test or Fisher's exact test, as appropriate. We used the Spearman coefficient to assess the correlation among the level of methylation of CpG sites, and to analyze the relationship among the SDS score. Logistic regression was used to examine the risk factors of preterm birth and SGA birth, the correlation between any two independent variables in the logistic regression equation should be less than 0.8. Skewed distribution data were log-transformed to obtain a normal distribution. Odds ratios with 95% CIs were calculated. All statistical analyses were performed with SPSS version 24.0, and a p-value < 0.05 was considered statistically significant.