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The aim of this study was to compare the clinical characteristics of singleton and twin pregnancies that resulted in spontaneous preterm births (sPTB) and to evaluate the prognostic value of phosphorylated insulin-like growth factor binding protein-1 (phIGFB-1) and placental alpha macroglobulin-1(PAMG-1) for sPTB prediction in symptomatic women. The study included 420 women and included two parts. Firstly, we performed a retrospective cohort study comparing pregnancy and neonatal outcomes in 170 women with singleton pregnancies and spontaneous preterm birth before 37 weeks of gestation with 150 women with twin pregnancies who delivered at the same gestational age. In order to obtain the link between clinical and biochemical predictors of preterm labor we organized the second part of the research. The second part was a prospective observational study in 100 women with singleton and twin pregnancies between 24 and 33 + 6 weeks of gestation and symptoms of threatening preterm labor and intact membranes. We assessed the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for phIGFBP-1 and PAMG-1 in terms of sPTB within 7 days and 14 days after testing. The rate of preterm premature rupture of membranes was higher in singleton pregnancies (67.1 versus 42.8%,  p  = 0.034). Cervical shortening in multiples was diagnosed at an earlier mean gestational age than in singleton pregnancies (30.1 ± 4.3 versus 35.9 ± 3.1 weeks,  p  = 0.013). In the singleton pregnancies group, the rate of microbial cervical colonization and the rate of bacterial vaginosis were significantly higher than in twin pregnancies (49.4 versus 15.3%,  p  &lt; 0.001; 32.9 versus 12.0%,  p  = 0.007, respectively). Premature twins had a longer oxygen dependency period, while the singletons were more predisposed to infectious morbidity. The study showed low sensitivity of phIGFBP-1 for sPTB, while the sensitivity of the PAMG-1 test was higher (sensitivity 60% and 90% within 7 days after testing and 50 versus 75% within 14 days after testing). Both tests showed a high NPV for sPTB (93.3% for phIGFBP-1 versus 98.9% for PAMG-1). The NPV for preterm labor in twins was also high for both tests (93% for phIGFBP-1 and 96% for PAMG-1). Cervical shortening is the main risk factor of sPTB in women with twin pregnancies; sPTB among singletons is associated with ascendent infection, involving fetal membranes. The PAMG-1 test showed high PPV and NPV for sPTB in symptomatic women and could be a reliable prognostic tool in clinical obstetrics. High NPV was observed for phIGFBP-1 and PAMG-1 in twin pregnancies.

Clinical and biochemical markers of spontaneous preterm birth in singleton and multiple pregnancies