Gestational Age

To revise the growth chart, thorough literature searches were performed to find published and unpublished population-based preterm size at birth (weight, length, and/or head circumference) references. The inclusion criteria, defined a priori, designed to minimize bias by restriction [13 ], were to locate population-based studies of preterm fetal growth, from developed countries with:
a) Corrected gestational ages through fetal ultrasound and/or infant assessment and/or statistical correction;
b) Data percentiles at 24 weeks gestational age or lower;
c) Sample of at least 25,000 babies, with more than 500 infants aged less than 30 weeks;
d) Separate data on females and males;
e) Data available numerically in published form or from authors,
f) Data collected within the past 25 years (1987 to 2012) to account for any secular trends.

The effect of different background correction methods on reproducibility was assessed using data from 20 pairs of duplicate samples that were part of a previously published study of methylation in 891 infant whole blood samples (12 (link)). As part of this study, duplicate samples were located on separate 96 well plates that underwent independent bisulfite conversion, hybridization and array scanning. One sample was excluded due to poor data quality, leaving 19 duplicate pairs (38 samples) for evaluation.
The effect of different background correction methods on measurement accuracy was assessed using data from methylation control mixture samples for this same study (12 (link)), where purified human 100% methylated and unmethylated DNA (Zymo Research, Irving CA) were mixed together in different proportions to create laboratory control samples with specific methylation levels: 0%, 5%, 10%, 20%, 40%, 50%, 60%, 80% and 100% methylated Replicates for each methylation level (n = 10, 3, 2, 3, 3, 2, 3, 3 and 10, respectively) were independently assayed on different arrays.
To avoid possible impact on evaluations, we excluded 69 075 probes, which include non-specific bind probes, common (MAF > 0.05) SNPs at CpG target regions, probes on sex chromosomes and probes with multimodal methylation distributions identified using ENmix R package. We also excluded probes with low quality methylation values where the number of beads was less than 3 or detection P-value greater than 0.05.
To demonstrate the effect of ENmix background correction method on epigenome-wide association studies (EWAS), we re-analyzed raw blood DNA methylation data from 889 infants in relation to maternal smoking (12 (link)). We preprocessed the data with different methods or combinations of methods: raw data, Q5 background correction, ENmix_oob background correction, ENmix and dye bias correction (ENmixD), ENmix+dye bias correction+quantile normalization (ENmixDQ) and ENmix+dye bias correction+quantile normalization+BMIQ (ENmixDQB). We used a robust linear regression model to test for association between maternal smoking and infant DNA methylation level adjusting for the following variables: cell type proportion (CD8T, CD4T, NK, Bcell, Mono and Gran) estimated using the Houseman method (13 (link)) from minfi R package, gestational age in weeks, sex, education in two categories, birth weight, maternal age, maternal BMI, parity, experimental batch, cleft phenotype and baby birth year.

We conducted a retrospective cohort study of all women readmitted to Meir Medical Center, from January 2014 through March 2020, with new delayed-onset of postpartum preeclampsia. Delayed-onset postpartum preeclampsia was defined as a new diagnosis of preeclampsia that occurred 48 h to 6 weeks postpartum. Preeclampsia was defined according to the American College of Obstetricians and Gynecologists criteria as blood pressure of 140 mm Hg systolic or 90 mm Hg diastolic or higher on two or more occasions more than 6 h apart, accompanied by proteinuria or end organ dysfunction, or blood pressure 160 mm Hg systolic or 110 mmHg diastolic or higher [9 (link)]. Excluded from the study women with prior diagnosis of preeclampsia, gestational hypertension, or chronic hypertension as well as women with prior chronic diseases.
The control group included randomly recruited healthy parturients with uncomplicated pregnancies, who came, during 2020, to the hospital for a routine screening hearing test for their newborns in the neonatal clinic, during postpartum period, on days 2–11.
.Data were collected by electronic medical record review and included: maternal age, gravity and parity, characteristics of current pregnancy (gestational age at delivery, mode of delivery, neonatal birth weight) and hemoglobin level on the day of labor. The postpartum hospitalization data included postpartum day of readmission and clinical features on presentation including pulse rate (beat per minute, bpm), blood pressure and serum laboratory values of liver function and platelet count. Pulse rate (bpm) and blood pressure of the control group were measured after at least 10 min of rest in a sitting position.
The study was approved by the Meir Medical Center Institutional Review Board on 17^th March 2020, number MMC-0048–20. All methods were carried out in accordance with relevant guidelines and regulations. Informed consent was not obtained from subjects due to the study nature.

Preterm birth was the primary outcome of this study, which was defined as births before 37 completed weeks of gestation. The World Health Organization (WHO) further subdivided preterm birth based on gestational age: extremely preterm (< 28 weeks), very preterm (28 to < 32 weeks), and moderate or late preterm (32 to < 37 weeks) [23 (link)]. Secondary outcomes were NICU admission, low birthweight and small for gestational age. Low birthweight was defined as a birthweight < 2500 g, and small for gestational age was defined as a birthweight less than the 10th percentile. The following variables were collected: maternal age at delivery (years), race [Asian, Black (Black or African American), White, other (American Indian or Alaska Native, Native Hawaiian or Other Pacific Islander, and more than one race)], education [less than 12 grade, high school/general educational development (GED), some college or associate degree (AA), bachelor or higher], pre-pregnancy weight (lb), pre-pregnancy body mass index (BMI) (BMI < 18.5 kg/m², underweight; BMI = 18.5–24.9 kg/m², normal; BMI = 25.0–29.9 kg/m², overweight; BMI = 30.0–34.9 kg/m², obesity), delivery weight (lb), weight gain (lb), smoking before pregnancy (yes or no), smoking status 1st/2nd/3rd trimester (mother-reported smoking in the three trimesters of pregnancy, yes or no), hypertension eclampsia (yes or no), gestational hypertension (yes or no), pre-pregnancy hypertension (yes or no), number of prenatal visits, the Special Supplemental Nutrition Program for Women, Infants, and Children (WIC, receipt of WIC food for the mother during this pregnancy, yes or no), plurality, prior birth now living, prior birth now dead, prior other terminations, total birth order, gestational age (weeks), newborn sex (female or male), birth weight (g), infertility treatment used (yes or no), pregnancy method (natural pregnancy, pregnancy via ART), method of delivery [spontaneous, non-spontaneous (forceps, vacuum, cesarean)], preterm birth [extremely preterm, very preterm, moderate or late preterm; spontaneous, indicated (forceps, vacuum, cesarean)], NICU admission, low birthweight (yes or no), and small for gestational age (yes or no). WIC is a program intended to help low income pregnant women, infants, and children through age 5 receive proper nutrition by providing vouchers for food, nutrition counseling, health care screenings and referrals; it is administered by the U.S. Department of Agriculture (https://ftp.cdc.gov/pub/Health_Statistics/NCHS/Dataset_Documentation/DVS/natality/UserGuide2019-508.pdf). Infertility treatment referred to using fertility enhancing drugs, artificial insemination, intrauterine insemination, or using ART. ART included in vitro fertilization (IVF), gamete intrafallopian transfer (GIFT), and zygote intrafallopian transfer (ZIFT). Information on variables is available at https://www.cdc.gov/nchs/nvss/index.htm.

Continuous data were tested for normality using the Kolmogorov-Smirnov test, and the continuous data of normal distribution were described as mean ± standard deviation (Mean ± SD), and the t-test was used for comparisons between groups. Non-normally distributed continuous variables were shown by median and quartile [M (Q₁, Q₃)], and the Wilcoxon rank sum test was used for comparisons between groups. Categorical data of groups were compared with the Pearson’s χ² test, and expressed as cases and the constituent ratio [n (%)]. Statistical power was calculated (power = 1). Missing data were imputed using multiple imputation (Supplementary Table 1). Data before imputation were also used for multivariate analyses to conduct sensitivity analyses.
In order to study the association between GDM and preterm birth among vAMA women, we established three models, and odds ratios (ORs) with 95% confidence intervals (CIs) were estimated. Model 1 was a univariate model. Model 2 was a multivariate model adjusting for maternal age at delivery, race, education, and newborn sex. Then all variables were included in a multivariable model for stepwise regression, and the following variables were adjusted for in Model 3: maternal age at delivery, race, education, newborn sex, pre-pregnancy weight, pre-pregnancy BMI, delivery weight, weight gain, smoking before pregnancy, hypertension eclampsia, gestational hypertension, pre-pregnancy hypertension, number of prenatal visits, plurality, total birth order, prior birth now living, prior other terminations, birth weight, pregnancy method, and method of delivery. Subgroup analyses were then performed based on race and use of infertility treatment to demonstrate if and how the association between GDM and preterm birth in vAMA women varied by race and use of infertility treatment. Further, preterm birth was subdivided into extremely preterm, very preterm, and moderate or late preterm birth. Logistic regression was used to investigate the association between GDM and different stages of preterm birth. Model 1 was a univariate model. Model 2 was a multivariate model correcting for maternal age at delivery, race, education, and newborn sex. Model 3 was a multivariate model correcting for maternal age at delivery, race, education, newborn sex, delivery weight, smoking status 2nd trimester, hypertension eclampsia, gestational hypertension, pre-pregnancy hypertension, number of prenatal visits, WIC, plurality, prior other terminations, total birth order, birth weight, pregnancy method, and method of delivery. As for the associations of GDM with NICU admission, low birthweight and small for gestational age in vAMA women, analytical methods were the same as those for the association between GDM and preterm birth, and subgroup analyses by race and use of infertility treatment were also conducted for these outcomes.
All statistical analyses were two-sided, and P < 0.05 was considered to be statistically significant. All analyses were completed by SAS 9.4 software (SAS Institute Inc., Cary, NC, USA).