DNA extraction from placental tissue was carried out according to the technique by Sambrook et al. [16] with some modifications. The I/D polymorphism of the ACE gene was determined by a polymerase chain reaction (PCR) method. Amplification was carried out in a total volume 25 mL that contained 15 nM of each primer (ACE F: 5'-CTG GAG ACC ACT CCC ATC CTT TCT-3', ACE R: 5'-GAT GTG GCC ATC ACA TTC GTC AGA T-3'), 67 mM Tris-Hcl (pH 8.8), 16.6 mM of ammonium sulfate, 6.7 mM MgCl_{2, 6.7 mM EDTA, 10 mM mercaptoethanol, 170 mkg BSA, 1.0 mM of each dNTP and one1 of Taq-polymerase (‘Bion’, Moscow, Russia). The PCR was performed under the following conditions: denaturation at 94°C for 7 min., followed by 30 cycles of amplification at 94°C for 40 seconds, 60°C for 40 seconds, 72°C for 1 min. and 72°C for 5 min. The DNA fragments were separated in a 7% polyacrylamide gel and subsequently stained with ethidium bromide and visualized under ultraviolet light.}

A standard chi-square test was used for comparing the genotype and allele frequencies. The relative risk was estimated by odds ratio (OR) and p values of <0.05 were considered to be significant. All confidence intervals (CIs) were calculated at the 95% level. Statistical analysis of results was performed using ‘STATISTICA v5.5a’ software.