Detection and Quantification of Male-Specific Fetal DNA in the Serum of Pregnant Cynomolgus Monkeys (Macaca fascicularis)

Because of their developmental similarities to humans, nonhuman primates are often used as a model to study fetal development for potential clinical applications in humans. The detection of fetal DNA in maternal plasma or serum offers a source of fetal genetic material for prenatal diagnosis. However, no such data have been reported for cynomolgus monkeys (Macaca fascicularis), an important model in biomedical research. We have developed a specific, highly sensitive PCR system for detecting and quantifying male-specific fetal DNA in pregnant cynomolgus monkeys. We used multiplex quantitative real-time PCR to analyze cell-free DNA in maternal blood serum obtained from 46 pregnant monkeys at gestational weeks 5, 12, and 22. The presence of SRY gene and DYS14 Y chromosomal sequences was determined in 28 monkeys with male-bearing pregnancies. According to confirmation of fetal sex at birth, the probe and primers for detecting the Y chromosomal regions at each time point revealed 100% specificity of the PCR test and no false-positive or false-negative results. Increased levels of the SRY-specific sequences (mean, 4706 copies/mL serum DNA; range, 1731 to 12,625) and DYS14-specific sequences (mean, 54,814 copies/mL serum DNA; range, 4175–131,250 copies) were detected at week 22. The SRY- and DYS14-specific probes appear to be an effective combination of markers in a multiplex PCR system. To our knowledge, this report is the first to describe the detection of cell-free DNA in cynomolgus monkeys.Abbreviations: C_t, threshold cycleAnalysis of cell-free circulating nucleic acids in human maternal plasma or serum has led to the development of risk-free methods for prenatal genetic diagnosis and the assessment of several fetal and maternal conditions, for example, sex determination for paternally inherited diseases, pregnancy-associated complications, sex-linked disorders for ambiguous genitalia, and embryo tracking.^{1,4,12,14,18,19} Technical challenges associated with detecting fetal DNA arise due to the low concentration of fetal DNA in maternal plasma during pregnancy and the difficulty of differentiating the genetic material of the fetus from that of the mother.^5,13,20 Fetal sex determination using sequences derived from the Y chromosome only is relatively simple and has a reported accuracy rate in humans of approximately 99.0% at 7 wk of gestation and 100% after 20 wk, depending on the protocol and methods used.^3,5,17,20 In other species, researchers have used real-time PCR assays during pregnancy to predict fetal sex from cell-free DNA at an accuracy of 100%.^9,10,11 Cell-free fetal DNA in the maternal circulation represents only 3% to 6% of the total free DNA obtained from plasma throughout pregnancy; however, this percentage is variable between pregnancies.^5,13,20In clinical biomedical research, it is essential to develop animal models for human diseases to reveal their mechanisms.^16,22 Continued progress in surgical intervention and molecular medicine suggests that it may soon be possible to develop potential treatments or even cures for several fetal genetic diseases at an early stage of pregnancy.¹⁵ Fetal developmental research during early pregnancy might be facilitated by using cell-free fetal DNA in the maternal blood rather than other methods, such as serum screening and ultrasonography. Nonhuman primates, especially macaques, are useful model animals for studying fetal development because of the similarity of the reproductive characteristics, placental structure, and developmental events between these animals and humans.^9,10 These developmental similarities highlight the importance of the study of cell-free fetal DNA in nonhuman primates and its usefulness as a marker to obtain genetic information about the fetus.In the current study, we investigated the presence of cell-free fetal DNA in the maternal plasma of cynomolgus monkeys by developing and using a standardized PCR system. To this end, we selected the SRY (sex-determining region Y) gene and DYS14 sequences of the cynomolgus monkey to use as sex-associated markers. The Y chromosome-specific sequences in the single-copy sex determination region of SRY and the multicopy (thus yielding increased sensitivity) sequences of DYS14 in the TSPY (testis-specific protein, Y-linked) gene have had wide clinical use in humans as molecular markers for detecting and quantifying cell-free fetal DNA.^3,7 In addition, TSPY has been used in bovines for detecting cell-free fetal DNA² and in rhesus macaques for long-term evaluation of microchimerism.⁸ Given the reports of fetal sex determination in rhesus macaques^9,10 and sheep¹¹ by analyzing Y chromosome-specific sequences from cell-free DNA, we hypothesized that we could predict the fetal sex of cynomolgus monkeys at different stages of gestation. This information has been extremely useful in optimizing the design of experimental studies in biomedical research and in managing a nonhuman primate breeding colony.¹⁰ Because cynomolgus and rhesus macaques are closely related members of the same genus, the current experiments are similar to a previous study.⁹We developed an efficient 2-color multiplex PCR system to detect and quantify fetal DNA in the maternal serum of cynomolgus monkeys during pregnancy. We used 2 loci on the Y chromosome in a single PCR test to minimize the likelihood of false-positive signals. Here we report the results of detection and analysis of fetal DNA at various weeks of gestation and evaluate our PCR system for its ability to determine fetal sex from pregnant monkeys’ cell-free DNA.