A Low-Cost Efficient Multiplex PCR for Prenatal Sex Determination in Bovine Fetus Using Free Fetal DNA in Maternal Plasma

Invasive methods for prenatal diagnosis, include chorionic villus sampling (CVS) and amniocentesis that entail a risk of fetal loss and mortality. In 1997, Lo et al. (1) demonstrated the existence of fetal DNA in plasma and serum from healthy pregnant women. Recent studies have shown that fetal DNA in maternal plasma have a mean of 3.4% and 6.2% of total DNA in early and late gestation, respectively (2) and are cleared at an extremely rapid rate following birth (3). Fetal sex determination is now possible at 8 weeks of pregnancy, by testing maternal blood samples. The reasonable sensitivity in PCR techniques could be considered to detect small amounts of fetal DNA in maternal plasma, based on the identification of specific regions of X and Y chromosomes circulating in maternal blood. Recent technical advances enable us to use both intact fetal cells (4-7) and cell-free fetal DNA (8-10) in maternal plasma and serum for non-invasive fetal gender and also prenatal genetic identifications. However, amount of the fetal DNA that is obtained by these simple methods is not enough to reach the desired intention. Most of the technical improvements such as fluorescence-based polymerase chain reaction (PCR) (11) and real-time PCR (12-13) methods are highly sensitive and technically demanding. However, expensive equipment limits their application in a routine setting. Some conventional PCR analyses of maternal plasma, serum and blood using the Y-specific sequences for example; DYS14, (14) DYZ3 (15), DYZ1 (16), the Y-specific repeat sequences (17) and sex determination region Y (SRY) (18-20) have been introduced for the diagnosis of fetal sexing. But in a routine setting internal amplification control for examination of results is difficult to be interpreted (20). A synchronic amplification of the X-Y homologous region of the amelogenin in human (21) and bovine (22) is reported also, bovine zfx and zfy gene sequences in maternal blood using a pair primers have also been described for fetal sexing (23).

The aim of this study was to establish a rapid and reliable method for sexing of bovine fetuses.

This has prompted us to improve another non-invasive method of bovine fetal sex determination using multiplex PCR amplification of the X chromosome (467 bp amelogenin gene) and the Y chromosome (341 bp amelogenin gene fragments and BC1.2-sequence (that is derived from a male-specific bovine DNA sequences) simultaneously.

The discovery of cell-free fetal DNA in maternal plasma in 1997 has opened up new possibilities for non-invasive diagnoses (1). Many related studies in humans (25-26), monkeys (27) and bovine (28) have proved that this fetal DNA is produced from the mechanism of cell transfer in the conceptus. However, there were reports of successful prediction of fetal sex through amplification of the male specific sequences (SRY) from the blood of pregnant cows (28). Thus, we have studied amelogenin gene and BC1.2 male specific bovine DNA sequence amplification in the maternal blood of pregnant cows.

In this study, we developed a multiplex PCR system for prenatal identification of fetal sexing. Though PCR methods cannot be compared with real-time PCR approach (12, 13), conventional PCR techniques provide a more practical methodology with acceptable sensitivity and specificity. The simple and simultaneous amplification of amelogenin gene on the X and Y chromosomes and the BC1.2 male specific bovine DNA sequence on the Y chromosome in a multiplex PCR system, respectively, could provide a satisfactory result for prenatal fetal determination. Moreover, it would be possible to accurately identify the fetal gender using PCR analysis of 38 maternal plasma samples during 8-38 weeks of gestation (Fig1 B and Table 1). No false-positive or false-negatives were generated at all trimesters of pregnancy among 38 pregnant heifers. In all pregnant heifers, the final accuracy of %100 was observed.

Unlike other described conventional PCR systems for prenatal fetal sex determinations on maternal plasma (15-16), the incorporation of the X-specific amelogenin gene amplification as an internal control of the multiplex PCR system herein described could greatly improve the reliability of the fetal sex identification. The use of multiplex PCR approach described here would be more practical in any laboratory where a conventional PCR is available. The ease, speediness and efficiency shown by this multiplex approach which requires no further modification of routine PCR procedure or additional advanced equipment should be directly applicable to a non-invasive prenatal fetal sex prediction by maternal plasma.

Like the results of similar studies in human (13, 19-20) and bovine (28-29), our results demonstrated that our fetal sex determination method using plasma is practical.

One of the benefits of this study and similar studies (16) was DNA extraction using phenol-chloroform method that costs lower than using an extraction kit. In addition, DNA that is extracted using phenol-chloroform method has appropriate quality and quantity.

The results have shown that phenol-chloroform method is a simple and suitable method for isolation of fetal DNA in maternal blood. Furthermore, multiplex PCR technique is cost-efficient, reliable and available for non-invasive sex determination in bovine fetus.