This is a longitudinal, observational, multicenter study performed from January 2019 to January 2021. The study participants were recruited at the High Risk Maternity Mangiagalli Centre, Department of Woman, Child and Neonate, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Milan, and at the Unit of Obstetrics, Department of Obstetrics and Gynecology, MBBM Foundation at San Gerardo Hospital, Monza, Italy.
The objective of the study is to create a predictive model for the development of perinatal complications, using sFlt-1/PlGF values and without knowing the delivery gestational age.
All pregnant women admitted for HDP, FGR, or both, were eligible for the present study. Women with an uneventful pregnancy were recruited as control group. Exclusion criteria were: multiple pregnancies, genetic or structural anomalies of the fetus, maternal infections, maternal age less than 18 years old. All patients provided written, informed consent prior to enrolment. The protocol of the study complies with the European Union's Good Clinical Practice standards and the Declaration of Helsinki, and it was approved by the Milan Area 2 Ethics Committee (MATER n° 71_2020 Fondazione IRCCS) and PREBIO STUDY (San Gerardo Hospital).
Patients were recruited at the time of referral at the outpatient high risk clinics, or at admission at the Maternal Fetal Medicine Wards. The visit included a general and obstetrical physical examination, with the assessment of maternal weight gain and the measurement of blood pressure. The diagnosis of HDP was made according to ISSHP guidelines , including both gestational hypertension (GH) and chronic hypertension (CH). Patients were evaluated longitudinally during pregnancy and they all underwent an obstetric ultrasound in order to evaluate fetal well-being by the measurement of the fetal biometry, Amniotic Fluid Index (AFI), and Doppler velocimetry of uterine arteries (mean UtA PI), umbilical arteries (UA PI), middle cerebral artery (MCA PI) and the fetal ductus venosus (PIV) when appropriate. The diagnosis of FGR was made according to the Delphi consensus .
Placental biomarkers sFlt-1 and PlGF were measured at recruitment. Venous blood was sampled and collected in tubes containing a separating gel, then the tubes were labeled and centrifuged for 10 minutes, within three hours of collection. sFlt-1 and PlGF were analysed by Roche's Elecsys automated method, immunoassays based on electro-chemoluminescence technology. The limits of detection varied between 10 and 8500 pg/ml for sFlt-1 and between 3 and 10000 pg/ml for PlGF.
For the purpose of the study, patients were classified according to the occurrence of perinatal complications. We considered as perinatal complications: intrauterine fetal death (IUFD), neonatal death, preterm delivery (<34 weeks) on maternal indication, newborn’s pH <7.1 or BE >-12, Apgar score <7 at 5 minutes, neonatal respiratory distress syndrome (RDS), retinopathy of the premature (ROP), necrotizing enterocolitis (NEC), persistent ductus arteriosus, sepsis, acute renal failure, pneumothorax, and hypoxic-ischemic encephalopathy (HIE).
We chose to use the Mann Whitney U-test for scalar variables and Fisher's exact test for categorical variables. Data are expressed as mean and standard deviation for continuous variables and as absolute and relative frequencies for categorical, respectively.
The survey is divided into an initial interpretative analysis of the test population and a subsequent predictive phase.
The first step of our study was a Survival Analysis performed in order to evaluate perinatal complications in relation to gestational age at delivery. The event of interest is the gestational age at the onset of complications. Cumulative incidence curves were built using Kaplan Meier estimates and gestational age at delivery was considered as the time variable. We considered the cumulative incidence of perinatal complications distinguished for the four risk categories established on the basis of the sFlt-1/PlGF ratio values at recruitment (low <38, medium 38-85/38-110, high >85/>110 and very high >655/>201 before and after the 34th week of gestation respectively) . Log rank test was used to compare the curves.
The second step of our analysis was a logistic regression model in order to understand whether sFlt-1/PlGF ratio is among the predictors of perinatal complications and how it influences the outcome. The dependent variable of the model were perinatal complications. The covariates we used were: sFlt-1/PlGF ratio at recruitment, pre-pregnancy BMI, maternal age, aspirin (ASA) or low-molecular-weight heparin (LMWH) during pregnancy, gestational diabetes, mean UtA PI, pre-pregnancy disease, gestational age at recruitment, parity, previous PE/FGR/IUFD, conceive with assisted reproductive technology (ART), and the macro-ethnicity of women divided into North African, Central and South African, Asian, Indian and South American. Odds Ratio were used to interpret the relationship between predictors and perinatal complications, and Wald test was performed in order to assess the significance of predictors.
Finally, the third step consisted in a classification analysis for which we used a Random Forest (we specified 500 trees and 3 randomly selected variables for each split) to create a model capable of predicting the occurrence of these complications. The dataset was divided into two groups: 75% of the data were used to train the model (training set), and 25% of the data were used to test the model (test set). Furthermore, since the number of pregnancies with perinatal complications was much lower than uncomplicated pregnancies, we adopted the Random Over Sampling Examples (R.O.S.E.) algorithm to balance only the training set. Finally, the model for predicting perinatal complications built on the training set was applied to the test set and its classification performances were reported. Mean decrease Gini index was used to evaluate the rank of each variable. Statistical analysis was performed by a statistician using R version 4.0.3 (2020-10-10).