We applied a noninvasive prenatal test for the determination of fetal gender in multiple pregnancies by using free fetal DNA circulating in maternal blood in order to evaluate whether the quantification of male DNA could distinguish the fetal gender and the number of male and female fetuses in multiple pregnancies. We enrolled consecutively 44 women with twin pregnancies between 11–14 weeks of gestation. Peripheral maternal blood was collected, and genomic DNA was extracted from maternal plasma and analyzed for the multicopy DYS 14 sequence by using real-time PCR to quantify male DNA. Results showed that male DNA concentration was significantly higher in twin pregnancies with at least one male fetus, compared to twin pregnancies with only female fetuses. Comparing male DNA concentration in pregnancies with two male fetuses versus pregnancies with one female fetus and one male fetus, we did not obtain a significant difference between the two groups due to a slight overlapping of the range values. Therefore, our test correctly predicted fetal gender, distinguishing twin pregnancies with at least one male fetus from twin pregnancies with only female fetuses, with a diagnostic accuracy of 100%. For distinguishing pregnancies with two male fetuses from pregnancies with both female and male fetuses, a diagnostic accuracy of 76.1% was achieved.

In the present study 90 multiple pregnancies were examined. These were subdivided on the basis of the number of embryos involved (74 twins, 10 triplets, 6 quintuplets) and on whether they were followed at our clinic for the entire pregnancy or not. In each group we analysed certain variables, calculating the respective mean values and standard deviations. We used the ANOVA test to discriminate the eventual differences in the means of the variables analysed, operating a p<0.05 significance value. In addition, significant differences were analysed by the test of Contrasts (Scheffe F-test). The concept that emerged from the data investigated is that careful management of these pregnancies, carried out in high-level structures, can reduce the incidence of complications on both the maternal and fetal side and thus prevent “minimal brain damage” in the newborn.

Over the last decades the frequency of multiple births has been on the decline in most developed countries. This trend, however, has been restricted to dizygotic twins, while monozygotic rates have remained stable or risen slowly. In more recent years, however, the fall in multiple dizygotic birth rates has ceased and a slight increase is observed. This trend and the increased frequency of triplets or higher-order births, registered from the early 1980s onwards, are essentially related to treatments for infertility. No single risk factor, such as maternal age, parity, oral contraceptive use, or declining fertility rates can explain the overall declining trends. In this paper we review the descriptive epidemiology and the main risk factors for multiple pregnancies.